The kinase Isr1 negatively regulates hexosamine biosynthesis in S. cerevisiae

Emma B Alme; Erica Stevenson; Nevan J Krogan; Danielle L Swaney; David P Toczyski

doi:10.1371/journal.pgen.1008840

. 2020 Jun 24;16(6):e1008840. doi: 10.1371/journal.pgen.1008840

The kinase Isr1 negatively regulates hexosamine biosynthesis in S. cerevisiae

Emma B Alme ^1,², Erica Stevenson ^3,^4,⁵, Nevan J Krogan ^3,^4,⁵, Danielle L Swaney ^3,^4,⁵, David P Toczyski ^1,^2,^*

Editor: Jason MacGurn⁶

PMCID: PMC7340321 PMID: 32579556

Abstract

The S. cerevisiae ISR1 gene encodes a putative kinase with no ascribed function. Here, we show that Isr1 acts as a negative regulator of the highly-conserved hexosamine biosynthesis pathway (HBP), which converts glucose into uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the carbohydrate precursor to protein glycosylation, GPI-anchor formation, and chitin biosynthesis. Overexpression of ISR1 is lethal and, at lower levels, causes sensitivity to tunicamycin and resistance to calcofluor white, implying impaired protein glycosylation and reduced chitin deposition. Gfa1 is the first enzyme in the HBP and is conserved from bacteria and yeast to humans. The lethality caused by ISR1 overexpression is rescued by co-overexpression of GFA1 or exogenous glucosamine, which bypasses GFA1’s essential function. Gfa1 is phosphorylated in an Isr1-dependent fashion and mutation of Isr1-dependent sites ameliorates the lethality associated with ISR1 overexpression. Isr1 contains a phosphodegron that is phosphorylated by Pho85 and subsequently ubiquitinated by the SCF-Cdc4 complex, largely confining Isr1 protein levels to the time of bud emergence. Mutation of this phosphodegron stabilizes Isr1 and recapitulates the overexpression phenotypes. As Pho85 is a cell cycle and nutrient responsive kinase, this tight regulation of Isr1 may serve to dynamically regulate flux through the HBP and modulate how the cell’s energy resources are converted into structural carbohydrates in response to changing cellular needs.

Author summary

Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, integrating a variety of environmental signals to drive cellular growth. Yeast encode over 100 kinases, yet many remain poorly characterized. The S. cerevisiae gene ISR1 encodes a putative kinase with no ascribed function. Here, we show that Isr1 decreases the synthesis of a critical structural carbohydrate, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), by mediating inhibition of one of the enzymes responsible for its synthesis, Gfa1. UDP-GlcNAc is the precursor to protein glycosylation, GPI anchor formation, and chitin synthesis, the first two of which are essential and conserved in humans. Throughout the cell cycle, and in response to changing environmental conditions, the cell must balance its use of glucose for energy production and generation of these structural carbohydrates. Here we show that Isr1 is regulated by both cell cycle and nutrient changes, and is rapidly degraded in a phosphorylation dependent manner. Isr1-mediated inhibition of UDP-GlcNAc synthesis may serve as a mechanism of dynamically regulating how the cell utilizes glucose in response to its environment.

Introduction

Protein phosphorylation is a major signaling mechanism that is critical to the control of most cellular processes [1–3]. It is estimated that 75% of the proteome is phosphorylated and kinases are among the largest protein families, comprising about 2% of most eukaryotic genomes [4]. Kinases often act in cascades to amplify signals and there is significant cross-talk amongst protein kinases, allowing for the coordination of many cellular inputs. Despite their abundance and critical regulatory roles in the cell, many of the approximately 130 yeast kinases remain poorly characterized [5].

One such kinase is Inhibitor of Staurosporine Resistance 1 (Isr1). ISR1 was first identified as a gene whose deletion is synthetically lethal with a temperature-sensitive allele of PKC1 and whose overexpression sensitized cells to staurosporine, a broad spectrum kinase inhibitor that targets Pkc1 [6]. While the molecular basis for these phenotypes was unknown, they suggested a defect in cell wall homeostasis. Pkc1 is essential to the cell wall integrity pathway, which is critical to remodeling the cell wall in response to cellular growth and environmental stress [7]. Other studies identified ISR1 as a gene whose overexpression causes sensitivity to caffeine, a general cell wall stress agent, and resistance to zymocin [8]. As chitin is the cellular receptor for zymocin [9], these phenotypes are consistent with reduced levels of chitin, a minor but critical structural component of the cell wall, comprising approximately 2% of the cell wall by mass [10]. The major protein component of the cell wall is glycosylphosphatidylinositol (GPI) anchors. Both GPI anchors and chitin share the same carbohydrate precursor, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which is also the precursor for protein glycosylation [10]. Many proteins involved in cell wall carbohydrate biosynthesis are heavily glycosylated, making regulated production of this carbohydrate essential for proper cell wall homeostasis. N-glycosylation is also critical to many other cellular processes, including proper protein folding and trafficking as well as cell cycle progression [10,11]. The cellular requirement for UDP-GlcNAc changes throughout the cell cycle. While protein glycosylation occurs continuously as a cell grows, chitin synthesis predominates in G1 at the time of bud emergence and chitin deposition also greatly increases in response to cell wall stress [12–16].

UDP-GlcNAc is synthesized via the hexosamine biosynthesis pathway (HBP), which is highly conserved from bacteria to humans [17]. Most of the glucose imported into the cell is shunted into glycolysis after it is converted to fructose-6-phosphate, but approximately 6% enters the hexosamine biosynthesis pathway where it is converted into UDP-GlcNAc though the sequential action of four enzymes: glutamine: fructose-6-phosphate aminotransferase (Gfa1), GlcN-6-P acetyltransferase (Gna1), Phosphoacetyl-glucosamine mutase (Pcm1) and UDP-GlcNAc pyrophosphorylase (Qri1) [18]. The first step of this pathway, conversion of fructose-6-phosphate and glutamine to glucosamine-6-phosphate, is mediated by Gfa1 and is the rate limiting step of the pathway [13,17]. Gfa1 activity has been shown to be directly proportional to UDP-GlcNAc synthesis and is regulated by nutrient and stress-responsive kinases [19–23].

We have identified a role for Isr1 as an inhibitor of the HBP, establishing a function for this kinase. High levels of Isr1 promote Gfa1 phosphorylation, and inhibition of the HBP by Isr1 is relieved by mutation of these Gfa1 phosphosites or bypassing Gfa1 by exogenous addition of glucosamine. Additionally, we have identified a critical Pho85-regulated SCF^CDC4 phosphodegron in Isr1. Mutation of this phosphodegron strongly affects HBP activity, highlighting how strict control of this kinase by post-translational modification can allow for signal integration and tight regulation of the HBP in response to changing cellular stimuli.

Results

Overexpression of Isr1 kinase activity is lethal

To begin to understand the role Isr1 might play in the cell, we first sought to test the effect of overexpressing ISR1. We placed the ISR1 ORF under the control of the GAL1 promoter on a 2μ plasmid. At this level of expression (referred to as GAL-2μ), Isr1 is lethal to the cell (Fig 1A). Isr1 is annotated as a putative protein kinase due to an unusual catalytic motif, HGD, in place of the signature HRD motif in its kinase domain. Mutation of the predicted aspartic acid proton acceptor in this motif to alanine abolished the lethality of GAL-2μ ISR1, indicating that kinase activity is required for Isr1 function (Fig 1A). Isr1 is a very low abundant protein [24] and difficult to overexpress for reasons that are currently unknown. The expression level of Isr1 from a 2μ plasmid utilizing the ISR1 promoter (referred to as 2μ ISR1) is low compared to GAL-2μ ISR1, and even GAL-2μ ISR1 does not result in extremely high levels of Isr1 (Fig 1B). To confirm this, we empirically determined the levels of Isr1 achieved by the GAL- 2μ ISR1 construct. We epitope tagged Pkc1 expressed under its endogenous promoter using a 3x-Flag tag, the same epitope tag used for Isr1, and compared these proteins by western blot. This comparison demonstrates that GAL-2μ Isr1 protein levels are similar to the endogenous expression level of Pkc1 (Fig 1C).

Fig 1 — (A) Wild type cells were transformed with an empty vector (EV) or 2μ plasmid expressing either a wild-type (GAL-2μ *ISR1*) or a kinase-dead allele of *ISR1* (GAL-2μ *isr1-D280A)* from the *GAL1* promoter. Cells were serially diluted on YPD or YPGal plates. (B) Western blot showing relative expression level of Isr1-3xFlag expressed on a 2μ plasmid from either its endogenous promotor (2μ *ISR1*) or the *GAL1* promoter (GAL-2μ *ISR1*). Values represent the normalized relative band intensity for Isr1. PSTAIR is a monoclonal antibody that recognizes the PSTAIR sequence in Cdc28 and is shown as a loading control. Cells were inoculated into YM-1 with the indicated carbon source and grown for 4 hours under NAT selection. **(C)** Relative levels of Isr1-3xFlag expressed from the *GAL1* promoter compared to Pkc1-3xFlag tagged at the endogenous locus.

ISR1 does not function in RNA processing

In contrast to the lethality we saw with GAL-2μ ISR1, in large scale studies using the MATa deletion collection, deletion of ISR1 has resulted in few phenotypes [25]. ISR1 deletion was shown to result in sensitivity to cordycepin, an adenosine analogue that causes premature termination, indicating a role in RNA processing [26]. However, we found that while the isr1Δ::KanMX strain isolated from the MATa deletion collection did show sensitivity to cordycepin (Fig 2A), this was not rescued by integrating ISR1 at the endogenous locus in this strain. By contrast, we observed that isr1Δ was resistant to tunicamycin, an N-glycosylation inhibitor [27]. This phenotype was not previously reported and sensitivity to tunicamycin was restored by integrating ISR1 in the isr1Δ strain (Fig 2A). This indicates that tunicamycin resistance, but not cordycepin sensitivity, is dependent on Isr1 activity. The end of the ISR1 coding sequence overlaps with the 3’UTR of its neighboring gene, YTH1, which is an essential component of the mRNA cleavage and polyadenylation factor. Thus, the cordycepin sensitivity of the isr1Δ strain from the deletion collection is likely a result of a reduction in function of YTH1.

Fig 2 — (A) *isr1*Δ::*KanMX*, isolated from the *Mat a* deletion collection, exhibits a sensitivity to cordycepin that is not shared by *isr1*Δ::*HygMX* generated in this study. Both exhibit resistance to tunicamycin. Strains of the indicated genotypes were grown on YPD alone or containing 40 μg/ml cordycepin or 0.5 μg/ml tunicamycin. (B) 2μ *ISR1* causes sensitivity to tunicamycin and resistance to calcofluor white (CFW). Strains of the indicated genotypes were transformed with EV, or a 2μ plasmid expressing either a wild-type (2μ *ISR1*) or a kinase-dead allele of *ISR1* (2μ *isr1-D280A)* from the *ISR1* promoter. Strains were spotted on CSM in the presence or absence of 0.25 μg/ml tunicamycin or 30 μg/ml CFW. (C) Overexpression of *ISR1* does not cause sensitivity to DTT. Wild-type cells were transformed with GAL-2μ *ISR1* or GAL-2μ *isr1-D280A* and spotted as in B on CSM-URA plates containing glucose, galactose or galactose + 1 mM DTT. (D) The hexosamine biosynthesis pathway converts fructose-6-phosphate and glutamine into UDP-GlcNAc, the precursor to N-glycosylation, GPI-anchors, and chitin. Gln1 acts upstream of the HBP and is required for glutamine synthesis. (E) GAL-2μ *ISR1* is synthetically lethal with all the enzymes in the HBP. Diploid heterozygous deletions of the enzymes in the HBP were generated and transformed with GAL-2μ *ISR1* and struck on YPD or YPGal plates. Note that wild-type diploids are less sensitive than haploid strains to *ISR1* overexpression.

Importantly, many of the genetic interactions reported for ISR1 from large scale studies using the MATa deletion collection are likely due to this neighboring gene effect as well, making it difficult to discern which phenotypes are specific to ISR1. This caveat of large scale collections has been previously described and the neighboring gene effect is estimated to result in incorrect annotation of approximately 10% of genes [28]. We generated our own isr1Δ strain using a hygromycin selectable marker and found that this strain did not exhibit sensitivity to cordycepin, but did show resistance to tunicamycin (Fig 2A). We speculate that the HygMX sequence used here might function as a more stable 3’UTR for YTH1 than the KanMX sequence used to generate the isr1Δ strain in the deletion collection. This isr1Δ::HygMX strain is used in all subsequent experiments.

ISR1 overexpression has phenotypes associated with a deficiency in UDP-GlcNAc

Given the resistance to tunicamycin that we uncovered with our newly generated isr1Δ strain, we wished to determine if higher levels of ISR1, reciprocally, caused tunicamycin sensitivity. Because of the lethality associated with GAL-2μ ISR1, we performed this characterization utilizing 2μ ISR1. We added a NAT selectable marker to the plasmid to allow drug screening on rich media containing Nourseothricin (NAT) instead of synthetic dropout media, allowing for lower concentrations of drugs to be used. 2μ overexpression of ISR1, but not a kinase dead allele, isr1-D280A, resulted in sensitivity to tunicamycin (Fig 2B). Tunicamycin acts as an ER-stress agent by inhibiting the first step in N-glycosylation, which involves the transfer of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from UDP-N-acetylglucosamine (UDP-GlcNAc) to the carrier lipid dolichyl-phosphate (Dol-P) [27,29]. Thus, the sensitivity to tunicamycin in response to ISR1 overexpression could be due to a defect in N-glycosylation or a general increase in ER-stress. To distinguish between these possibilities, we tested if ISR1 overexpression conferred sensitivity to other ER stress agents and found that GAL-2μ ISR1 did not confer sensitivity to DTT, suggesting that the tunicamycin sensitivity is not due to a general increase in ER-stress (Fig 2C). For reasons that are currently unknown, the GAL-2μ ISR1 phenotype is less severe on synthetic media, allowing this sensitivity test to be performed.

We also found that 2μ ISR1 conferred resistance to calcofluor white (Fig 2B), recapitulating previous data indicating a role for ISR1 in cell wall integrity [8]. Calcofluor white is a cell-wall stress agent that targets chitin and resistance indicates a specific deficiency in chitin deposition in the cell wall. Deletion of the main chitin synthase, CHS3, results in complete resistance to calcofluor white, whereas other cell wall mutants or overproduction of chitin results in sensitivity [9,30–32].

Both chitin and protein glycosylation are produced from the same precursor carbohydrate, UDP-GlcNAc, which is also the precursor for GPI anchors (Fig 2D). Chitin and GPI anchors predominantly function at the cell surface: GPI anchors are the major protein component of the cell wall, whereas chitin is a more minor, but still critical structural component [10,14]. N-glycosylation functions on the cell surface as well, but is also essential for proper protein folding and trafficking [11]. Previous studies have shown that blocking GPI anchor formation increases chitin deposition in the cell wall [33]. UDP-GlcNAc synthesis is the rate-limiting step in the production of chitin, which is a polymer of UDP-GlcNAc, and GPI-anchor inhibition is thought to allow the accumulation of excess UDP-GlcNAc that is no longer converted into GPI anchors [13,33].

The tunicamycin sensitivity of the 2μ ISR1 strain and the tunicamycin resistance observed in the isr1Δ strain might be due to misregulation of protein N-glycosylation. If ISR1 overexpression specifically inhibited N-glycosylation, one would expect a larger pool of available UDP-GlcNAc for chitin synthesis, resulting in a compensatory increase in chitin production. However, we observed that 2μ ISR1 caused resistance to calcofluor white, indicating a deficiency in chitin deposition. This is consistent with a defect in both protein glycosylation and chitin biosynthesis. This suggest that Isr1 acts upstream of N-glycosylation, reducing UDP-GlcNAc production.

ISR1 overexpression is synthetically lethal with enzymes in the hexosamine biosynthesis pathway

Most of the glucose imported into the cell is shunted into glycolysis after it is converted to fructose-6-phosphate, but approximately 6% enters the hexosamine biosynthesis pathway (HBP), where fructose-6-phosphate and glutamine are converted into UDP-GlcNAc though the sequential action of four enzymes: Gfa1, Gna1, Pcm1, and Qri1 (Fig 2D) [18]. This pathway is highly conserved from bacteria to humans [17]. Given that each of these enzymes are essential, we generated heterozygote deletions in genes encoding each of these enzymes and expressed GAL1-ISR1 in these strains to test the genetic interactions of this pathway with ISR1. We found that GAL-2μ ISR1 is synthetically lethal with all four enzymes in the pathway (Fig 2E). Notably, diploid wild-type cells are less sensitive to GAL-2μ ISR1 as compared to haploid cells. Gln1 is not part of the HBP, but synthesizes glutamine, one of the precursors to the pathway [34], and also exhibits synthetic sickness in combination with GAL-2μ ISR1. These genetic interactions suggest that ISR1 overexpression inhibits the HBP.

ISR1 lethality is rescued by exogenous glucosamine, but not other precursors to UDP-GlcNAc synthesis

The first enzyme of this pathway, Gfa1, converts fructose-6-phosphate and glutamine to glucosamine-6-phosphate (Fig 3A) [35]. This is the rate-limiting step of the pathway and Gfa1 activity is proportional to chitin synthesis [13]. GFA1 essentiality can be bypassed by exogenous glucosamine, which can be phosphorylated by hexokinases. Thus, gfa1Δ strains are glucosamine auxotrophs. Addition of exogenous glucosamine drives flux through the HBP and increases UDP-GlcNAc production [35,36]. We tested if the lethality associated with GAL-2μ ISR1 overexpression can be rescued by addition of exogenous glucosamine and found that in the presence of 5mM glucosamine, GAL-2μ ISR1 no longer inhibited growth (Fig 3B). Some cell wall mutants have growth phenotypes that can be rescued by both exogenous glucosamine and by addition of sorbitol to the media [36]. As sorbitol is an osmotic stabilizer, this implies that the slow growth phenotype of such mutants is caused by cell lysis due to a weakened cell wall. To determine if a similar general cell wall defect is responsible for the lethality associated with GAL-2μ ISR1, we tested growth of this strain in the presence of 10% sorbitol and found that sorbitol was not capable of rescuing growth (S1 Fig). This suggests that the lethality of GAL-2μ ISR1 is due to a growth arrest caused by a deficiency of UDP-GlcNAc production and not cell lysis caused by a weakened cell wall.

As expected, addition of glucosamine also restored sensitivity of the 2μ ISR1 overexpression strain to calcofluor white by driving flux through the HBP and increasing the amount of UDP-GlcNAc (Fig 3C). By contrast, glucosamine did not restore sensitivity to chs3Δ (Fig 3C), as an increase in UDP-GlcNAc cannot restore chitin synthesis in the absence of the chitin synthase itself. Thus, the resistance to calcofluor white observed upon 2μ overexpression of ISR1 is unlikely to be due to a general trafficking defect that mislocalizes Chs3. This is in agreement with the lack of sensitivity observed upon exposure to DTT. Similarly, exogenous glucosamine restored growth to the 2μ ISR1 strain in the presence of tunicamycin (Fig 3C). The ability of glucosamine to rescue ISR1 overexpression phenotypes implies that Isr1 acts at or before the first step of the HBP. To determine if a deficiency in one of the precursors to the HBP is responsible for the ISR1 overexpression phenotypes, we tested if fructose and glutamine, the reactants in the production of glucosamine-6-phosphate (Fig 3A), could rescue the ISR1 overexpression phenotypes. We also tested alternative or non-fermentable carbon sources, which bypass glycolysis entirely, and found that neither the precursors to the HBP nor non-fermentable carbon sources were capable of rescuing ISR1 overexpression phenotypes (Fig 3C and 3D, S1 Fig). The ability of glucosamine, but not precursors to the HBP, to completely rescue ISR1 overexpression phenotypes indicates that Isr1 likely inhibits the first step of the HBP.

Isr1 is a negative regulator of Gfa1

Our observation that exogenous glucosamine rescues the lethality associated with GAL-2μ ISR1 overexpression suggests that Isr1 might inhibit flux through the HBP. If this is the case, overexpression of GFA1 should rescue this lethality as well. To explore this possibility, we co-overexpressed GFA1 on the same plasmid as GAL1-ISR1 and as expected, GFA1 overexpression also rescued the lethality associated with GAL-2μ ISR1 overexpression (Fig 4A). To determine if this was unique to GFA1, we co-overexpressed another enzyme downstream in this pathway, Qri1, with GAL1-ISR1. QRI1 only mildly increased growth on galactose, suggesting that Isr1 acts at the level of Gfa1 within the HBP (Fig 4A).

Fig 4 — (A) *GFA1* overexpression rescues the lethality of GAL-2μ *ISR1*. Wild-type cells were transformed with a 2μ plasmid expressing either *ISR1* or *isr1-D280A* from the GAL1 promoter and either *GFA1* or *QRI1* from their endogenous promoters. Cells were serial diluted onto YPD or YPGal. (B) The Gfa1 S332 S334 phosphopeptide is enriched in cells expressing GAL-2μ *ISR1*. Volcano plot of phosphopeptides detected in *isr1*Δ cells expressing EV or GAL-2μ *ISR1*. Peptides with a p < 0.01 and having a fold change +/- 4-fold different between are considered significant. (C) Gfa1 S332 is phosphorylated in an Isr1-dependent manner. Targeted data extraction for Gfa1 S332 phosphorylation sites, which was only detected in the Gal-2u *ISR1* conditions. P-value is the result of a two-sided unpaired t-test. (D) Mutation of *GFA1* phosphorylation sites at the endogenous locus ameliorates the lethality of GAL-2μ *ISR1*. Wild-type cells or a *GFA1* phosphomutant (S332A,T334A, S336A) were transformed with EV or GAL-2μ *ISR1* and spotted as in A. (E) The *GFA1-3A* allele is hypomorphic. Cells of the indicated genotypes were transformed with EV or 2μ *ISR1* and serial diluted on YPD in the presence or absence of 0.1 μg/ml tunicamycin. (F) Experiment was performed as in D with diploid cells of the indicated genotypes.

In other organisms, including C. albicans, drosophila, and humans, phosphorylation by multiple kinases, including AMPK and PKA, has been shown to both positively and negatively regulate Gfa1 activity [19,20,22,23,37,38]. Overexpression of ISR1 did not alter Gfa1 protein levels (S2 Fig), suggesting that it might act by inhibiting Gfa1 function. However, the GFA1-TAP allele was unable to support viability and therefore it remains possible that the epitope tagged GFA1 allele is stabilized or cannot be targeted by Isr1.

A previous mass spectrometry study of kinase interactors identified Gfa1 as a physical interactor with Isr1 in three separate immunoprecipitation experiments [39]. Given this, we sought to determine if Gfa1 is phosphorylated in an Isr1-dependent manner. We conducted phosphoproteomics in isr1Δ cells expressing either an empty vector or GAL-2μ ISR1 and detected over 8,779 distinct phosphorylated peptides on 1,860 proteins (S1 Table). In this experiment, we detected 4 sites of phosphorylation on Gfa1: S199, S253, S332, and T334. Of these sites, the doubly phosphorylated peptide containing S332 and T334 was 12.9-fold up-regulated in ISR1-expressing cells (Fig 4B). Additionally, a singly phosphorylated S332 peptide was also observed only in the ISR1-expressing cells. Targeted data extraction for this peptide revealed its phosphorylation level was 179-fold higher in ISR1-expressing cells (Fig 4C). To examine the importance of these phosphorylations, we mutated S332, T334, and S336 to alanine. S336 was included because it was adjacent to the other two sites and was seen to be phosphorylated in a previous whole-phosphoproteome screen [40]. Mutation of S332, T334, and S336 to alanine at the endogenous locus (referred to as GFA1-3A) rescued the lethality associated with GAL-2μ ISR1 overexpression (Fig 4D). This suggests that Isr1 inhibits Gfa1 by promoting its phosphorylation.

Notably, the GFA1-3A mutant was slightly hypomorphic. While the GFA1-3A strain had no growth defect under normal conditions, it was sensitive to tunicamycin and resistant to calcofluor white (Fig 4E, S2C Fig). This sensitivity was recessive, as expected for a hypomorph, and was epistatic to 2μ ISR1 (Fig 4D, S2 Fig). By contrast, the GFA1-3A mutant was also dominant in its ability to rescue GAL-2μ ISR1-induced lethality: the GFA1/GFA1-3A heterozygous diploid was resistant to GAL-2μ ISR1 (Fig 4F). Our finding that the resistance conferred by GFA1-3A is dominant is consistent with the hypothesis that GFA1-3A rescues ISR1 overexpression because it is refractory to inhibition by Isr1. Thus, while GFA1-3A may have slightly less activity than wild type in the uninhibited/unphosphorylated state, this still likely represents significantly more activity than the phosphorylated state. These data suggest Isr1 is a negative regulator of Gfa1.

It should be noted that, while the GFA1 phosphomutant rescued GAL-2μ ISR1-induced lethality, GFA1-3A cells overexpressing ISR1 still grew more slowly than GFA1-3A cells overexpressing isr1-D280A. Therefore, while our data support a model where Isr1 inhibits the HBP by promoting the phosphorylation of Gfa1, Isr1 may have additional substrates, additional sites on Gfa1, or both.

Isr1 is an unstable cell-cycle regulated protein targeted by Pho85 and Cdc4

Given that even mild overexpression of Isr1 inhibits the essential function of Gfa1, we next sought to understand how Isr1 is regulated in the cell. The cellular need for UDP-GlcNAc varies throughout the cell cycle and we therefore tested if Isr1 is a cell cycle regulated protein. Analysis of Isr1 protein levels after arresting cells in each phase of the cell cycle showed that Isr1 protein levels are largely confined to the G1/S transition and are drastically reduced in G1 and M phases (Fig 5A). This matches known transcriptional data for ISR1 [41]. Previous work from our lab suggested that Isr1 is a substrate of Cdc4 [42]. Cdc4 is an F box protein that functions as a substrate adaptor for the SCF ubiquitin ligase complex and targets many substrates for proteasome-mediate degradation in a cell-cycle dependent manner [43]. Consistent with this, a cycloheximide chase of Isr1-13xMyc in wild-type cells, as compared to temperature-sensitive mutants of CDC4 and CDC53, showed that Isr1 is unstable, with a half-life of approximately 30 minutes, and is stabilized by inactivation of CDC4 or CDC53 (Fig 5B).

Fig 5 — (A) Isr1 is cell cycle regulated. Western blot showing levels of Isr1-13xmyc in cells growing asynchronously or arrested in G1, S, or M phase with alpha factor, Hydroxyurea (HU) or nocodazole, respectively. PSTAIR and Pds1 are shown as loading and cell cycle arrest controls, respectively. PSTAIR is a monoclonal antibody that recognizes the PSTAIR sequence in Cdc28. In all panels, values represent the normalized relative band intensity for Isr1. (B) Isr1 is targeted for degradation by Cdc4. Cycloheximide-chase assay of Isr1-13xmyc in wild-type, *cdc4-1*, or *cdc53-1* strains. Cells were shifted to the non-permissive temperature for 30 minutes before addition of cycloheximide for the indicated number of minutes. Levels of Isr1-13xMyc and PSTAIR (loading control) are shown. (C) Degradation of Isr1 is dependent on Pho85. Experiment was performed as in B at 30°C in *PHO85* or *pho85*Δ cells. Rad53 is shown as a loading control (D) Isr1 is stabilized by deletion of *PCL1*. Experiment as in B at 30°C in *PCL1* or *pcl1*Δ cells. (E) Western blot showing levels of Isr1-13xMyc and PSTAIR (loading control) after shifting cells to low phosphate media for 60 minutes.

Cdc4 recognizes and binds a phosphodegron with the optimal sequence (S/T)-P-X2-4 –(S/T) in which both (S/T) are phosphorylated, often by CDK kinases [44–47]. Isr1 was previously identified as an in vitro substrate of Pho85 [48], a CDK-like kinase that can function with any of 10 different cyclins in response to changes in nutrient and cell-cycle conditions [49,50]. Isr1 was shown to be phosphorylated by Pho85 in complex with Pcl1, which functions in G1 progression [48,51]. We found that deletion of PHO85 stabilized Isr1, suggesting that Pho85-phosphorylation of Isr1 enables Isr1 to be targeted by Cdc4 for degradation (Fig 5C). Deletion of PCL1 also partially stabilized Isr1 (Fig 5D). Given this partial stabilization, it remains possible that other Pcls, in addition to Pcl1, can also function with Pho85 to target Isr1. Pho85 is inhibited by the CDK inhibitor Pho81 in low phosphate conditions [52]. Upon release into low phosphate media, Isr1 levels increased, as would be expected if Pho85 activity promotes its instability (Fig 5E). Pho85 activity has also been shown to regulate carbohydrate metabolism [49,53]. We therefore tested if Isr1 stability was altered by carbon source. We found that growth in glycerol, a non-fermentable carbon source, as well as glucose withdrawal, also partially stabilized Isr1, whereas growth in galactose did not (S3 Fig). This supports a model where Pho85-mediated degradation of Isr1 might allow Isr1 activity to be responsive to environmental conditions.

Stabilization of an endogenous Isr1 phosphodegron recapitulates Isr1 overexpression phenotypes

Examination of the Isr1 protein sequence revealed two putative Cdc4 phosphodegrons in the N-terminus of the protein, each comprising two CDK consensus phosphorylation sites separated by several amino acids (Fig 6A). We constructed an Isr1 phosphodegron mutant (ISR1-PD) by mutating these four residues (T4, S8, T86, S92) as well as two adjacent serine/threonine residues to alanine at the endogenous locus. The ISR1-PD mutant was completely stable (Fig 6B), strongly supporting a model where phosphorylation of Isr1 at these sites targets it for degradation by Cdc4.

Fig 6 — (A) Diagram of Pho85 and Cdc4 consensus sites in Isr1 that comprise a phosphodegron. Six sites (T3, T4, S8, T85, T86, S92) are mutated to alanine at the endogenous locus in the *ISR1-PD* mutant. (B) An Isr1 phosphodegron mutant is stable. Cycloheximide-chase assay of Isr1-13xmyc in wild-type, *pho85*Δ, or cells expressing *ISR1-PD*. Cycloheximide was added for the indicated number of minutes. Levels of Isr1-13xMyc and PSTAIR (loading control) are shown. PSTAIR is a monoclonal antibody that recognizes the PSTAIR sequence in Cdc28. (C) An Isr1 phosphodegron mutant is resistant to calcofluor white (CFW). Strains of the indicated genotypes were diluted onto YPD with or without 40 μg/ml CFW. (D) An Isr1 phosphodegron mutant is sensitive to tunicamycin. Experiment was performed as in C, except strains were spotted on YPD with or without 0.25 μg/ml tunicamycin. (E) Diploid strains of the indicated genotypes were diluted onto YPD with or without 0.25 μg/ml tunicamycin.

We next tested if this regulation is functionally relevant by examining the calcofluor white sensitivity of the ISR1-PD mutant. We found that these mutations are sufficient to cause resistance to calcofluor white, consistent with there being higher levels of Isr1 activity in ISR1-PD mutants (Fig 6C). This same resistance was recapitulated on Congo Red, a cell-wall stress agent that also targets chitin (S4 Fig) [54]. Similarly, the ISR1-PD mutant was also highly sensitive to tunicamycin (Fig 6D). Taken together, these data show that the endogenous phosphodegron mutant of Isr1 recapitulates ISR1 overexpression phenotypes, supporting a model where tight regulation of Isr1 is critical for controlling its function in the cell. Interestingly, deletion of the entire N-terminal 93 amino acids of Isr1, which includes both phosphodegrons, was also sensitive to tunicamycin (Fig 6D). This implies that this truncation mutant is active and the only function of the N-terminus of Isr1 is in mediating its instability. Our mass spectrometry data also identified an additional proline-directed phosphorylation site in Isr1, S47, that may also be targeted by Pho85 or could represent autophosphorylation.

To determine the physiological relevance of Isr1 regulation, we tested the genetic interactions of this Isr1 phosphodegron mutant with deletions of the enzymes of the HBP pathway. Because these genes are essential, we examined diploids that were heterozygous for each HBP gene, as well as the ISR1-PD mutant. We found that ISR1/ISR1-PD showed a level of sensitivity to tunicamycin similar to that seen for GFA1/gfa1Δ (Fig 6E). One interpretation of this is that one copy of the stabilized Isr1 phosphodegron mutant reduces Gfa1 activity by half. Notably, The GFA1/gfa1Δ strain grew more slowly on tunicamycin than deletions of the other three enzymes in the pathway, as would be expected given that Gfa1 is the rate-limiting step of the pathway. While ISR1/ISR1-PD and GFA1/gfa1Δ were virtually synthetically lethal in the presence of tunicamycin, this synthetic sickness was not observed when ISR1-PD was combined with other deletions in the pathway. The specificity of this synthetic interaction further supports a model where Isr1 activity negatively regulates Gfa1 function and emphasizes the large effect of an even minor increase in Isr1 stabilization on flux through the HBP.

Discussion

Throughout the cell cycle and in response to changing nutrient conditions, the cell must balance its utilization of glucose for energy needs and as a precursor to structural and signaling carbohydrates. Here, we uncover a function for a poorly characterized kinase, Isr1, as a negative regulator of the hexosamine biosynthesis pathway (HBP) that synthesizes UDP-GlcNAc from glucose.

Only ~15% of yeast genes are phenotypic when overexpressed [55]. The lethality of overexpressing ISR1, combined with the limited phenotypes of its deletion, support a model where Isr1 is a negative regulator of an essential protein. Our data firmly establishes a role for Isr1 in inhibition of the HBP based on several lines of evidence. First, 2μ overexpression of ISR1 results in resistance to calcofluor white and sensitivity to tunicamycin, whereas isr1Δ is slightly sensitive to calcofluor white and resistant to tunicamycin. These opposing phenotypes are consistent with a deficiency in UDP-GlcNAc when ISR1 is overexpressed and an increase in UDP-GlcNAc when ISR1 is absent. Genes that function in protein trafficking or the downstream utilization of UDP-GlcNAc in protein glycosylation are expected to have a compensatory increase in chitin production and to have a sensitivity to other ER-stress agents [33]. This was not observed for ISR1 overexpression, suggesting that it inhibits the HBP itself. Consistent with this, we found that ISR1 overexpression is synthetically lethal with every enzyme in the HBP.

All phenotypes of ISR1 overexpression are rescued by either overexpressing GFA1, the enzyme of the first step of the HBP, or by bypassing GFA1 function by the addition of exogenous glucosamine. Addition of reactants upstream of Gfa1, such as fructose and glutamine, had no effect. This strongly suggested that Isr1 inhibits this first step in the HBP. Furthermore, we found that phosphorylation of a specific serine in Gfa1 is dependent on Isr1 activity and mutation of this phosphorylation site to alanine rendered cells insensitive to ISR1 overexpression. Thus, Isr1 appears to act as a negative regulator of Gfa1. The GFA1 phosphomutant is hypomorphic, yet epistatic to ISR1 overexpression and dominant in its ability to rescue GAL-2μ ISR1 lethality, strongly implying that Isr1 exerts its function in the cell by promoting Gfa1 phosphorylation.

Isr1 may phosphorylate Gfa1 directly, as suggested by the existing immunoprecipitation-mass spectrometry data, or it might act in a kinase cascade. However, our phosphoproteomics dataset did not reveal specific activation of other kinases that might act downstream of Isr1 and deletion of several stress-responsive kinases did not rescue ISR1 lethality (S5 Fig). Notably, deletion of the majority of the N-terminal domain of Isr1 rendered Isr1 more active, implying that this portion of the protein is not required for substrate specificity. Given that the remaining residues comprise little more than a kinase domain, it remains unclear how Isr1 targets its substrates.

ISR1 was previously implicated in cell wall homeostasis by Mehlgarten et al.’s observation that ISR1 overexpression results in zymocin resistance, a cell wall stress agent [8]. The findings of that paper suggested a role for Isr1 in chitin biology and are in agreement with the function we have uncovered for Isr1 as a negative regulator of Gfa1. Mehlgarten et al. delete PHO85 and find this abolishes the ISR1 overexpression phenotype, leading the authors to posit that Pho85 activates Isr1. However, our results are directly contrary to that hypothesis and we suggest that the rescue that Mehlgarten et al. observed is due to the pleiotropic effects that Δpho85 has on the cell wall.

Additional genetic evidence suggests an interaction between ISR1 and GFA1: Isr1 was first identified due to the fact that its overexpression resulted in heightened sensitivity to staurosporine, an inhibitor of Pkc1, and ISR1 deletion is synthetically lethal with a temperature sensitive allele of PKC1 [6]. Pkc1 is known to positively regulate GFA1 transcription through Rlm1 activation [21,56]. Therefore, the synthetic lethality of ISR1 and PKC1 is consistent with a role for Isr1 as a negative regulator of Gfa1. Similarly, ISR1 has been reported in multiple studies to have strong negative genetic interaction with PRR1 [3,57,58], a kinase that is a physical interactor with Gfa1 [39]. These data would be consistent with Prr1 and Isr1 independently regulating Gfa1. Notably, prr1Δ does not rescue GAL-2μ ISR1 lethality (S5 Fig).

The human homologue of Gfa1, Gfat1, has been previously shown to be negatively regulated by phosphorylation via AMPK and PKA [19,23,37]. Thus, inhibition of Gfa1 via phosphorylation may be a convergent or conserved mechanism of regulating hexosamine biosynthesis in response to changes in nutrients. The cellular requirement for UDP-GlcNAc changes throughout the cell cycle. While protein glycosylation occurs continuously as a cell grows, chitin synthesis predominates in G1 at the time of bud emergence [12]. Indeed, Gfa1 expression increases during G1 or in response to pheromone or cell wall stress [21,33,41]. Thus, dynamic control of Gfa1 is required to properly shunt glucose into or away from the hexosamine biosynthesis pathway as needed. Notably, Isr1 protein levels peak at the G1/S transition, just after the period of time during the cell cycle at which higher levels of chitin synthesis are required. Given that Gfa1 activity is directly proportional to chitin synthesis [13], Isr1 might provide a mechanism of rapidly returning the cell to a normal level of chitin synthesis following bud emergence. Gfa1 is a highly abundant protein, whereas Isr1 is expressed at very low levels. We speculate that tight regulation of Isr1 protein levels via Pho85 and Cdc4 might allow for rapid dynamic changes in Gfa1 activity and could provide the cell with a less energetically costly mechanism of regulating Gfa1 function than degrading Gfa1 itself. In this way, Isr1 could function to integrate environmental signals to regulate the remodeling of the cell wall and glycoprotein biosynthesis. While more studies are required to determine the specific environmental inputs that modulate Isr1 instability and the mechanism by which Isr1 inhibits Gfa1, this work firmly establish a cellular function for Isr1 as a key negative regulator of an essential, conserved pathway.

Materials and methods

Strains and plasmids

Strains and plasmids used in this study are listed in S2 and S3 Tables, respectively. All yeast strains in this study are in the S288C background. Unless otherwise noted, single gene deletions are from the Mat a deletion collection (Open Biosystems) and TAP-tagged strains were a gift from Erin O’Shea and Jonathan Weismann. Cloning of constructs and transformations were done using standard techniques. Diploid heterozygous deletions were made by transformation of a KanMX cassette with homology to the promoter and 3’ UTR of the gene of interest into a wild type diploid or heterozygous ISR1-PD diploid strain. The ISR1-PD and GFA1-3A strains were constructed by gene replacement: point mutants were synthesized on a plasmid containing a nourseothricin (NAT) (ISR1) or HygMX (GFA1) selection marker (S2 Table, S6 Fig). A PCR fragment was generated with homology to the endogenous promoter at the 5’ end and the MX cassette at the 3’ end. This PCR fragment was transformed into the deletion strain to replace the gene deletion previously marked by HygMX (ISR1) or KanMX (GFA1). Integrations were screened by marker loss/gain and then verified by PCR. In the case of GFA1, GFA1-3A was first generated in a GFA1/gfa1Δ diploid and haploid mutants were isolated by tetrad analysis.

Yeast cell culture

Selective media lacking specific amino acids or nucleobases was made using Complete Supplement Mixture (CSM) from Sunrise Sciences, supplemented with yeast nitrogen base and 2% dextrose. Unless otherwise noted, cells were cultured at 30°C on YM-1 media supplemented with 2% dextrose or CSM-URA to maintain plasmid selection. 100 μg/ml NAT was added to maintain plasmids when strains were grown in YM-1. Expression from the GAL1 promoter was induced with 2% (wt/vol) galactose for 4 hours. For experiments with temperature-sensitive strains, cells were maintained at 23 °C and shifted to 37°C for 30 minutes before initiating the experiment. Low phosphate media was made as with CSM, but using YNB lacking amino acids and phosphate (Formedium CYN0803) and supplemented with 0.55g KCl/liter. To measure Isr1 protein levels in low phosphate, cells were washed several times in low-phosphate media and resuspended in low-phosphate media for 60 minutes.

Western blotting

From cultures in midlog phase, cell pellets of equivalent optical densities were collected, washed with 1 mL 4°C H₂O, and frozen on dry ice. Standard TCA precipitations were preformed to extract proteins. Samples were resuspended in SDS-PAGE sample buffer, boiled for 5 min, and cell lysates were analyzed by SDS-PAGE using 4–20% gradient Tis-HCl gels (BioRad, #3450034). Proteins were transferred onto 0.2-μm nitrocellulose membrane. Western blots were performed with low-salt PBS with Tween-20 (PBS-T) (15 mM NaCl, 1.3 mM NaH₂PO₄, 5.4 mM Na₂HPO₄, 0.05% Tween-20). Primary antibody incubations were performed in 5% (wt/vol) nonfat dry milk and low-salt PBS-T. Antibodies were used as follows: α-Rad53 (Abcam ab104232); α-Flag (Sigma-Aldrich, F3165); α-Myc (BioLegend, #626802), α-PSTAIR (Sigma-Aldrich P7962), α-Pds1 (generous gifts from Adam Rudner), α-TAP (Thermo-Scientific CAB1001). Western blots were visualized by the LiCor Odyssey Imaging System. Isr1 levels were quantified using the Image Studio Lite software and intensity values were normalized to the loading control for each lane.

Cell cycle arrest

Cells were grown overnight in YM-1 and inoculated to OD600 = 0.2 and grown at 30°C for 45 minutes. For arrest in G1, 15 μg/ml alpha factor was added. After 90 minutes, an additional 10 μg/ml alpha factor was added. Cells were harvested after 2.5 hours. For arrest in S and M phase respectively, 200 mM HU or 15 μg/ml nocodazole was added and cells were harvested after 2 hours. Arrest was confirmed by microscopic analysis and equivalent ODs were processed for western blots as described above.

Cycloheximide-chase assays

Cells were grown as for western blotting to midlog phase. Cycloheximide was added to cultures for a final concentration of 50 μg/ml after collection at the t = 0 time point. Equivalent ODs were collected for each time point and were processed for western blots as described above. For cdc53-1 and cdc4-1 experiments, cells (including wild-type control) were shifted to 37 °C for 30 minutes before addition of cycloheximide. For alternative carbon sources, cells were grown overnight in YM-1 supplemented with 2% dextrose, 2% galactose or 2% glycerol/1% ethanol, inoculated at 0.2 OD, and grown to mid-log phase before addition of cycloheximide.

Mass spectrometry sample preparation

Cells were grown overnight in YM-1 supplemented with 2% raffinose and 100 μg/mL NAT and then inoculated at 0.3 OD. After 1 hour, 40% galactose was added to a final concentration of 2% galactose and cells were grown for an additional 3 hours to OD ~0.8. 40 OD of cells were washed 2x with water and flash frozen in liquid nitrogen. Cells pellets were combined and lysed in a denaturing urea buffer (8 m urea, 0.1 m ammonium bicarbonate, pH 8, 150 mm NaCl, 1 Roche mini protease inhibitor tablet without EDTA/10 ml, ½ Roche phosSTOP tablet/10 ml) using 14 × 1.5 min bursts on a BioSpec mini bead-beater at room temperature. 2 ml screw-cap tubes used for lysis were pierced with an 18-gauge needle and spun in a swinging bucket centrifuge for 30 s at 1000 × g to collect extract. Extract was rotated end-over-end for 30 min at room temperature before clarification via centrifugation at 17,000 × g for 7 min followed by a second centrifugation at 17,000 × g for 2 min, both at room temperature. Extracts were quantitated using a BCA protein quantification kit (Pierce). 1 m TCEP (Sigma C4706–2) was added to final concentration of 4 mm to 1 mg of protein and incubated for 30 min at room temperature. 0.5 m iodoacetamide (Sigma L1149–5G, prepared fresh in water) was added to a final concentration of 10 mm and incubated in the dark for 30 min. To quench excess iodoacetamide, 0.5 m DTT was added to a final concentration of 10 mm for another 30 min in the dark. Samples were diluted ∼fourfold (to less than 2 m urea) with 0.1 m Tris, pH 8, and Lys-C/trypsin (Promega, Madison, WI, V5071, dissolved in 50 mm acetic acid) was added at a ratio of 200 μg trypsin to 1 mg total protein. Samples (∼1 ml total volume of diluted sample) were incubated for 20 hours at room temperature with rotation. After digestion, TFA was added to a final concentration of 0.3–0.1% TFA, with pH of final solution ~2.

Phosphopeptide enrichment by immobilized metal affinity chromatography

Iron nitrilotriacetic acid (NTA) resin were prepared in-house by stripping metal ions from nickel nitrilotriacetic acid agarose resin (Qiagen) with 100 mM ethylenediaminetetraacetic (EDTA) acid, pH 8.0 three times. Resin was washed twice with water and 100 mM iron(III) chloride was applied three times. The iron-NTA resin was washed twice with water and once with 0.5% formic acid. Iron-NTA beads were resuspended in water to create a 25% resin slurry. 60 μl of Fe-NTA resin slurry was transferred to individual Silica C18 MicroSpin columns (The Nest Group) pre-equilibrated with 100 μl of 80% ACN, 0.1% TFA on a vacuum manifold. Subsequent steps were performed with the Fe-NTA resin loaded on the Silica C18 columns. Peptide samples were mixed twice with the Fe-NTA resin and allowed to incubate for 2 minutes. The resin was rinsed four times with 200 μl of 80% ACN, 0.1% TFA. In order to equilibrate the chromatography columns, 200 μl of 0.5% formic acid was applied twice to the resin and columns. Peptides were eluted from the resin onto the C18 column by application of 200 μl of 500 mM potassium phosphate, pH 7.0. Peptides were washed twice with 200 μl of 0.5% formic acid. The C18 columns were removed from the vacuum manifold and eluted twice by centrifugation at 1000g with 60 μl of 50% ACN, 0.25% TFA. Peptides were dried with a centrifugal adaptor and stored at -20°C until analysis by liquid chromatograph and mass spectrometry.

Proteomic data acquisition and analysis

Peptides were resuspended in 45 uL of 4% formic acid, 3% ACN, and 2uL was analyzed in on a Bruke timsTOF Pro mass spectrometry system equipped with a Bruker nanoElute high-pressure liquid chromatography system interfaced via a captiveSpray source. Samples were directly injected on a C18 reverse phase column (25 cm x 75 μm packed with ReprosilPur C18 AQ 1.9 um particles). Peptides were separated by an organic gradient from 2 to 28% ACN at 400nl/min over the course of a 120 acquisition. Each sample was injected twice, once with data-dependent PASEF acquisition to build a spectral library, and one via a diaPASEF acquisition for quantitative analysis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [59] partner repository with the dataset identifier PXD018429. All data-dependent PASEF files [60] were search against the Saccharomyces cerevisiae proteome database (Downloaded from the Saccharomyces Genome Database January 13, 2015). Peptide and protein identification searches were performed with the Spectronaut Pulsar software (www.spectronaut.org) to generate a spectral library of detected phosphorylated peptides with a 1% false-discovery rate at the peptide and protein level. Spectronaut was further used with the default settings to analyzed the diaPASEF data [61] and extract quantitative regulation of detected phosphorylation sites. Label-free quantification and statistical testing of phosphorylation sites was performed using the artMS R-package (version 1.3.7) (https://github.com/biodavidjm/artMS), and the MSstats statistical R-package (version 3.16.0) [62]. Instant Clue [63] was used for figure generation of proteomics data.

Spot tests

Yeast strains were inoculated into 3–5 ml YM-1 or CSM-URA + 2% dextrose grown overnight with aeration at 30°. Fivefold dilution series were set up in 96-well plates, and 3–5 μl aliquots of the dilution series were transferred to YPD, YPGAL or CSM plates. Drug concentrations and alternative carbon sources are specified in individual figures. In experiments utilizing plasmids overexpressing ISR1, plates also contained 100 μg/ml NAT to maintain plasmid selection. Plates were incubated 2–3 days at 30° until colonies formed and then were photographed.

Supporting information

S1 Fig. ISR1 lethality is not rescued by osmotic stabilizers or other carbon sources.

(A) Lethality of GAL-2μ ISR1 is not rescued by sorbitol. Cells of the indicated genotypes expressing EV or GAL-2μ ISR1 were diluted onto YPD or YPGal + 10% sorbitol. (B) Lethality from GAL-2μ ISR1 overexpression is not rescued by alternative carbon sources. Wild-type cells were transformed with EV, GAL- 2μ ISR1 or GAL- 2μ isr1-D280A and spotted onto CSM containing 2% glucose, galactose or the indicated carbon source. (C) Experiment performed as in B, but with 3% glycerol.

(TIF)

Click here for additional data file.^{(2.5MB, tif)}

S2 Fig. ISR1 does not affect Gfa1 protein levels.

(A). 2μ ISR1 does not affect Gfa1 protein levels at any cell cycle stage. Cells were inoculated in CSM-URA and arrested in G1, S, and M phase with alpha factor, HU or nocodazole respectively. Note that Gfa1 was upregulated in response to alpha factor, as expected. (B) GAL-2μ ISR1 does not alter Gfa1 protein levels. Cells were grown overnight in CSM-URA raffinose and inoculated in CSM-URA galactose for 4 hours. (C) Cells of the indicated genotypes were transformed with EV or 2μ ISR1 and serial diluted on YPD with or without 30 μg/ml calcofluor white. (D) Diploids of the indicated genotypes were transformed with EV or 2μ ISR1 and spotted on YPD with or without 0.25 μg/ml tunicamycin.

(TIF)

Click here for additional data file.^{(3.1MB, tif)}

S3 Fig. Isr1 is partially stabilized in non-fermentable carbon sources.

(A) Isr1 is partially stabilized by glycerol/ethanol, but not galactose. Cycloheximide-chase assay of Isr1-13xmyc grown in YM-1 containing 2% dextrose, 2% galactose or 2% glycerol/1% ethanol. Cells were grown overnight in the indicated carbon source, inoculated in fresh media, and cycloheximide was added for the indicated number of minutes. For the no glucose condition, cells were grown in YM-1 with dextrose, washed twice in media without a carbon source, and suspended in media with no carbon source at the same time as adding cycloheximide.

(TIF)

Click here for additional data file.^{(478KB, tif)}

S4 Fig. Mutation of an Isr1 phosphodegron confers resistance to Congo Red.

(A) An Isr1 phosphodegron mutant is resistant to Congo Red. Strains of the indicated genotypes were diluted onto YPD in the presence or absence of 150 μg/ml Congo Red.

(TIF)

Click here for additional data file.^{(696.2KB, tif)}

S5 Fig. MAPKs and PRR1 are not downstream of ISR1.

(A) Strains of the indicated genotypes expressing Gal- 2μ ISR1 or GAL-2μ isr1-D280A (wild-type only) were struck on YPD or YPGal plates. (B) ppr1Δ cells expressing EV or GALl-2μ ISR1 were serial diluted onto YPD or YPGal.

(TIF)

Click here for additional data file.^{(2MB, tif)}

S6 Fig. ISR1 phosphodegron mutant sequence.

Double-stranded DNA sequence used to generate ISR1 phosphodegron mutant. Blue sequence is the end of the ISR1 promoter. Lowercase red base pairs indicate mutated residues.

(DOCX)

Click here for additional data file.^{(112.7KB, docx)}

S1 Table. Phosphoproteomics dataset for isr1Δ vs GAL-2μ ISR1.

Phosphopeptide dataset comparing isr1Δ cells expressing either EV or GAL-2μ ISR1. The "Results" tab contains the full set of quantitative results displayed in the volcano plot (Fig 4B). The "S332” tab contains the quantitative data displayed in Fig 4C.

(XLSX)

Click here for additional data file.^{(743.3KB, xlsx)}

S2 Table. Strains used in this study.

(DOCX)

Click here for additional data file.^{(120.9KB, docx)}

S3 Table. Plasmids used in this study.

(DOCX)

Click here for additional data file.^{(84.1KB, docx)}

Acknowledgments

We thank Jonathan Asfaha and David Morgan for reagents and generously allowing use of their equipment. We’d also like to thank the members of the Toczyski lab for helpful discussions.

Data Availability

All relevant data are within the manuscript and its Supporting Information files. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD018429.

Funding Statement

This work was supported by National Institutes of Health grants R35GM118104 to DPT and R01GM133981 to DLS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Ptacek J, Devgan G, Michaud G, Zhu H, Zhu X, Fasolo J, et al. Global analysis of protein phosphorylation in yeast. Nature. 2005;438: 679–684. 10.1038/nature04187 [DOI] [PubMed] [Google Scholar]
2.Manning G, Plowman GD, Hunter T, Sudarsanam S. Evolution of protein kinase signaling from yeast to man. Trends Biochem Sci. 2002;27: 514–520. 10.1016/s0968-0004(02)02179-5 [DOI] [PubMed] [Google Scholar]
3.Fiedler D, Braberg H, Mehta M, Chechik G, Cagney G, Mukherjee P, et al. Functional Organization of the S. cerevisiae Phosphorylation Network. Cell. 2009;136: 952–963. 10.1016/j.cell.2008.12.039 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Sharma K, D’Souza RCJ, Tyanova S, Schaab C, Wiśniewski JR, Cox J, et al. Ultradeep Human Phosphoproteome Reveals a Distinct Regulatory Nature of Tyr and Ser/Thr-Based Signaling. Cell Rep. 2014;8: 1583–1594. 10.1016/j.celrep.2014.07.036 [DOI] [PubMed] [Google Scholar]
5.Kannan N, Taylor SS, Zhai Y, Venter JC, Manning G. Structural and Functional Diversity of the Microbial Kinome. PLOS Biol. 2007;5: e17 10.1371/journal.pbio.0050017 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Miyahara K, Hirata D, Miyakawa T. Functional Interaction of Isr1, a Predicted Protein Kinase, with the Pkc1 Pathway in Saccharomyces cerevisiae. Biosci Biotechnol Biochem. 1998;62: 1376–1380. 10.1271/bbb.62.1376 [DOI] [PubMed] [Google Scholar]
7.Levin DE. Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway. Genetics. 2011;189: 1145–1175. 10.1534/genetics.111.128264 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Mehlgarten C, Zink S, Rutter J, Schaffrath R. Dosage suppression of the Kluyveromyces lactis zymocin by Saccharomyces cerevisiae ISR1 and UGP1. FEMS Yeast Res. 2007;7: 722–730. 10.1111/j.1567-1364.2007.00216.x [DOI] [PubMed] [Google Scholar]
9.Jablonowski D, Fichtner L, Martin VJ, Klassen R, Meinhardt F, Stark MJR, et al. Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor. Yeast. 2001;18: 1285–1299. 10.1002/yea.776 [DOI] [PubMed] [Google Scholar]
10.Orlean P. Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall. Genetics. 2012;192: 775–818. 10.1534/genetics.112.144485 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Larkin A, Imperiali B. The Expanding Horizons of Asparagine-Linked Glycosylation. Biochemistry. 2011;50: 4411–4426. 10.1021/bi200346n [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Schekman R, Brawley V. Localized deposition of chitin on the yeast cell surface in response to mating pheromone. Proc Natl Acad Sci U S A. 1979;76: 645–649. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC383005/ [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Lagorce A, Berre-Anton VL, Aguilar-Uscanga B, Martin-Yken H, Dagkessamanskaia A, François J. Involvement of GFA1, which encodes glutamine–fructose-6-phosphate amidotransferase, in the activation of the chitin synthesis pathway in response to cell-wall defects in Saccharomyces cerevisiae. Eur J Biochem. 2002;269: 1697–1707. 10.1046/j.1432-1327.2002.02814.x [DOI] [PubMed] [Google Scholar]
14.Lesage G, Bussey H. Cell Wall Assembly in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2006;70: 317–343. 10.1128/MMBR.00038-05 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Bulik DA, Olczak M, Lucero HA, Osmond BC, Robbins PW, Specht CA. Chitin Synthesis in Saccharomyces cerevisiae in Response to Supplementation of Growth Medium with Glucosamine and Cell Wall Stress. Eukaryot Cell. 2003;2: 886–900. 10.1128/ec.2.5.886-900.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Valdivia RH, Schekman R. The yeasts Rho1p and Pkc1p regulate the transport of chitin synthase III (Chs3p) from internal stores to the plasma membrane. Proc Natl Acad Sci. 2003;100: 10287 10.1073/pnas.1834246100 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Milewski S. Glucosamine-6-phosphate synthase—the multi-facets enzyme. Biochim Biophys Acta BBA—Protein Struct Mol Enzymol. 2002;1597: 173–192. 10.1016/S0167-4838(02)00318-7 [DOI] [PubMed] [Google Scholar]
18.Milewski S, Gabriel I, Olchowy J. Enzymes of UDP-GlcNAc biosynthesis in yeast. Yeast. 2006;23: 1–14. 10.1002/yea.1337 [DOI] [PubMed] [Google Scholar]
19.Zibrova D, Vandermoere F, Göransson O, Peggie M, Mariño KV, Knierim A, et al. GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis. Biochem J. 2017;474: 983–1001. 10.1042/BCJ20160980 [DOI] [PubMed] [Google Scholar]
20.Graack HR, Cinque U, Kress H. Functional regulation of glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1) of Drosophila melanogaster in a UDP-N-acetylglucosamine and cAMP-dependent manner. Biochem J. 2001;360: 401–412. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1222241/ [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Zheng J, Khalil M, Cannon JF. Glc7p Protein Phosphatase Inhibits Expression of Glutamine-Fructose-6-phosphate Transaminase from GFA1. J Biol Chem. 2000;275: 18070–18078. 10.1074/jbc.M000918200 [DOI] [PubMed] [Google Scholar]
22.Milewski S, Kuszczak D, Jedrzejczak R, Smith RJ, Brown AJP, Gooday GW. Oligomeric Structure and Regulation of Candida albicans Glucosamine-6-phosphate Synthase. J Biol Chem. 1999;274: 4000–4008. 10.1074/jbc.274.7.4000 [DOI] [PubMed] [Google Scholar]
23.Chang Q, Su K, Baker JR, Yang X, Paterson AJ, Kudlow JE. Phosphorylation of Human Glutamine:Fructose-6-phosphate Amidotransferase by cAMP-dependent Protein Kinase at Serine 205 Blocks the Enzyme Activity. J Biol Chem. 2000;275: 21981–21987. 10.1074/jbc.M001049200 [DOI] [PubMed] [Google Scholar]
24.Ghaemmaghami S, Huh W-K, Bower K, Howson RW, Belle A, Dephoure N, et al. Global analysis of protein expression in yeast. Nature. 2003;425: 737–741. 10.1038/nature02046 [DOI] [PubMed] [Google Scholar]
25.Giaever G, Chu AM, Ni L, Connelly C, Riles L, Véronneau S, et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature. 2002;418: 387–391. 10.1038/nature00935 [DOI] [PubMed] [Google Scholar]
26.Holbein S, Wengi A, Decourty L, Freimoser FM, Jacquier A, Dichtl B. Cordycepin interferes with 3’ end formation in yeast independently of its potential to terminate RNA chain elongation. RNA. 2009;15: 837–849. 10.1261/rna.1458909 [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Yoo J, Mashalidis EH, Kuk ACY, Yamamoto K, Kaeser B, Ichikawa S, et al. GlcNAc-1-P-transferase-tunicamycin complex structure reveals basis for inhibition of N-glycosylation. Nat Struct Mol Biol. 2018;25: 217–224. 10.1038/s41594-018-0031-y [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Ben-Shitrit T, Yosef N, Shemesh K, Sharan R, Ruppin E, Kupiec M. Systematic identification of gene annotation errors in the widely used yeast mutation collections. Nat Methods. 2012;9: 373–378. 10.1038/nmeth.1890 [DOI] [PubMed] [Google Scholar]
29.Barnes G, Hansen WJ, Holcomb CL, Rine J. Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step. Mol Cell Biol. 1984;4: 2381–2388. 10.1128/mcb.4.11.2381 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Ram AFJ, Klis FM. Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red. Nat Protoc. 2006;1: 2253–2256. 10.1038/nprot.2006.397 [DOI] [PubMed] [Google Scholar]
31.Ram AFJ, Wolters A, Hoopen RT, Klis FM. A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast. 1994;10: 1019–1030. 10.1002/yea.320100804 [DOI] [PubMed] [Google Scholar]
32.Roncero C, Valdivieso MH, Ribas JC, Durán A. Isolation and characterization of Saccharomyces cerevisiae mutants resistant to Calcofluor white. J Bacteriol. 1988;170: 1950–1954. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC211056/ [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Sobering AK, Watanabe R, Romeo MJ, Yan BC, Specht CA, Orlean P, et al. Yeast Ras Regulates the Complex that Catalyzes the First Step in GPI-Anchor Biosynthesis at the ER. Cell. 2004;117: 637–648. 10.1016/j.cell.2004.05.003 [DOI] [PubMed] [Google Scholar]
34.Mitchell AP. The GLN1 Locus of SACCHAROMYCES CEREVISIAE Encodes Glutamine Synthetase. Genetics. 1985;111: 243–258. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1202641/ [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Watzele G, Tanner W. Cloning of the glutamine:fructose-6-phosphate amidotransferase gene from yeast. Pheromonal regulation of its transcription. J Biol Chem. 1989;264: 8753–8758. Available: http://www.jbc.org/content/264/15/8753 [PubMed] [Google Scholar]
36.Gomez A, Perez J, Reyes A, Duran A, Roncero C. Slt2 and Rim101 Contribute Independently to the Correct Assembly of the Chitin Ring at the Budding Yeast Neck in Saccharomyces cerevisiae. Eukaryot Cell. 2009;8: 1449–1459. 10.1128/EC.00153-09 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Eguchi S, Oshiro N, Miyamoto T, Yoshino K, Okamoto S, Ono T, et al. AMP-activated protein kinase phosphorylates glutamine : fructose-6-phosphate amidotransferase 1 at Ser243 to modulate its enzymatic activity. Genes Cells. 2009;14: 179–189. 10.1111/j.1365-2443.2008.01260.x [DOI] [PubMed] [Google Scholar]
38.Frisa PS, Sonneborn DR. Developmentally regulated interconversions between end product-inhibitable and noninhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by protein dephosphorylation-phosphorylation reactions. Proc Natl Acad Sci. 1982;79: 6289–6293. 10.1073/pnas.79.20.6289 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Breitkreutz A, Choi H, Sharom JR, Boucher L, Neduva V, Larsen B, et al. A Global Protein Kinase and Phosphatase Interaction Network in Yeast. Science. 2010;328: 1043–1046. 10.1126/science.1176495 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Holt LJ, Tuch BB, Villén J, Johnson AD, Gygi SP, Morgan DO. Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution. Science. 2009;325: 1682 10.1126/science.1172867 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Spellman PT, Sherlock G, Zhang MQ, Iyer VR, Anders K, Eisen MB, et al. Comprehensive Identification of Cell Cycle–regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray Hybridization. Mol Biol Cell. 1998;9: 3273–3297. 10.1091/mbc.9.12.3273 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Mark KG, Simonetta M, Maiolica A, Seller CA, Toczyski DP. Ubiquitin Ligase Trapping Identifies an SCFSaf1 Pathway Targeting Unprocessed Vacuolar/Lysosomal Proteins. Mol Cell. 2014;53: 148–161. 10.1016/j.molcel.2013.12.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Willems AR, Schwab M, Tyers M. A hitchhiker’s guide to the cullin ubiquitin ligases: SCF and its kin. Biochim Biophys Acta BBA—Mol Cell Res. 2004;1695: 133–170. 10.1016/j.bbamcr.2004.09.027 [DOI] [PubMed] [Google Scholar]
44.Tang X, Orlicky S, Liu Q, Willems A, Sicheri F, Tyers M. Genome-Wide Surveys for Phosphorylation-Dependent Substrates of SCF Ubiquitin Ligases. Methods in Enzymology. Academic Press; 2005. pp. 433–458. 10.1016/S0076-6879(05)99030-7 [DOI] [PubMed] [Google Scholar]
45.Skowyra D, Craig KL, Tyers M, Elledge SJ, Harper JW. F-Box Proteins Are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex. Cell. 1997;91: 209–219. 10.1016/s0092-8674(00)80403-1 [DOI] [PubMed] [Google Scholar]
46.Hao B, Oehlmann S, Sowa ME, Harper JW, Pavletich NP. Structure of a Fbw7-Skp1-Cyclin E Complex: Multisite-Phosphorylated Substrate Recognition by SCF Ubiquitin Ligases. Mol Cell. 2007;26: 131–143. 10.1016/j.molcel.2007.02.022 [DOI] [PubMed] [Google Scholar]
47.Lyons NA, Fonslow BR, Diedrich JK, Yates JR, Morgan DO. Sequential Primed Kinases Create a Damage-Responsive Phosphodegron on Eco1. Nat Struct Mol Biol. 2013;20: 194–201. 10.1038/nsmb.2478 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Dephoure N, Howson RW, Blethrow JD, Shokat KM, O’Shea EK. Combining chemical genetics and proteomics to identify protein kinase substrates. Proc Natl Acad Sci. 2005;102: 17940–17945. 10.1073/pnas.0509080102 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Carroll AS, Bishop AC, DeRisi JL, Shokat KM, O’Shea EK. Chemical inhibition of the Pho85 cyclin-dependent kinase reveals a role in the environmental stress response. Proc Natl Acad Sci U S A. 2001;98: 12578–12583. 10.1073/pnas.211195798 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Huang D, Friesen H, Andrews B. Pho85, a multifunctional cyclin-dependent protein kinase in budding yeast. Mol Microbiol. 2007;66: 303–314. 10.1111/j.1365-2958.2007.05914.x [DOI] [PubMed] [Google Scholar]
51.Espinoza FH, Ogas J, Herskowitz I, Morgan DO. Cell cycle control by a complex of the cyclin HCS26 (PCL1) and the kinase PHO85. Science. 1994;266: 1388–1391. 10.1126/science.7973730 [DOI] [PubMed] [Google Scholar]
52.Schneider KR, Smith RL, O’Shea EK. Phosphate-regulated inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81. Science. 1994;266: 122–126. 10.1126/science.7939631 [DOI] [PubMed] [Google Scholar]
53.Huang D, Moffat J, Wilson WA, Moore L, Cheng C, Roach PJ, et al. Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10. Mol Cell Biol. 1998;18: 3289–3299. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC108910/ [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Imai K, Noda Y, Adachi H, Yoda K. A Novel Endoplasmic Reticulum Membrane Protein Rcr1 Regulates Chitin Deposition in the Cell Wall of Saccharomyces cerevisiae. J Biol Chem. 2005;280: 8275–8284. 10.1074/jbc.M409428200 [DOI] [PubMed] [Google Scholar]
55.Sopko R, Huang D, Preston N, Chua G, Papp B, Kafadar K, et al. Mapping Pathways and Phenotypes by Systematic Gene Overexpression. Mol Cell. 2006;21: 319–330. 10.1016/j.molcel.2005.12.011 [DOI] [PubMed] [Google Scholar]
56.Levin DE. Cell Wall Integrity Signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2005;69: 262–291. 10.1128/MMBR.69.2.262-291.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Bandyopadhyay S, Mehta M, Kuo D, Sung M-K, Chuang R, Jaehnig EJ, et al. Rewiring of Genetic Networks in Response to DNA Damage. Science. 2010;330: 1385–1389. 10.1126/science.1195618 [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Costanzo M, VanderSluis B, Koch EN, Baryshnikova A, Pons C, Tan G, et al. A global genetic interaction network maps a wiring diagram of cellular function. Science. 2016;353: aaf1420 10.1126/science.aaf1420 [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ, et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 2019;47: D442–D450. 10.1093/nar/gky1106 [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Meier F, Brunner A-D, Koch S, Koch H, Lubeck M, Krause M, et al. Online Parallel Accumulation–Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer. Mol Cell Proteomics MCP. 2018;17: 2534–2545. 10.1074/mcp.TIR118.000900 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Meier F, Brunner A-D, Frank M, Ha A, Voytik E, Kaspar-Schoenefeld S, et al. Parallel accumulation–serial fragmentation combined with data-independent acquisition (diaPASEF): Bottom-up proteomics with near optimal ion usage. bioRxiv. 2019; 656207 10.1101/656207 [DOI] [PubMed] [Google Scholar]
62.Choi M, Chang C-Y, Clough T, Broudy D, Killeen T, MacLean B, et al. MSstats: an R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments. Bioinforma Oxf Engl. 2014;30: 2524–2526. 10.1093/bioinformatics/btu305 [DOI] [PubMed] [Google Scholar]
63.Nolte H, MacVicar TD, Tellkamp F, Krüger M. Instant Clue: A Software Suite for Interactive Data Visualization and Analysis. Sci Rep. 2018;8 10.1038/s41598-018-31154-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS Genet. doi: 10.1371/journal.pgen.1008840.r001

Decision Letter 0

Gregory P Copenhaver, Jason MacGurn

7 Apr 2020

* Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. *

Dear Dr Toczyski,

Thank you very much for submitting your Research Article entitled 'The kinase Isr1 negatively regulates hexosamine biosynthesis in S. cerevisiae' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

All three reviewers have had the opportunity to read and review your submission, and there is consensus that this is a strong paper that communicates important findings that will be of interest to those studying cell signaling pathways and the regulation of cellular metabolism. We also agree with reviewers that several minor issues need to be addressed prior to publication. However we believe this can be addressed with changes to the text of the manuscript, and will not require additional experimentation. Some of the issues raised that you should address in the revised manuscript include:

1. Reviewer 1 points out some inaccuracies in the literature, surrounding the evolutionary relationship between ISR1 and Raf. Considering the points made by Reviewer 1, the authors are advised to make significant changes to the introduction and discussion of the manuscript in order to address these concerns. (See Reviewer 1, point #1 and point #5.)

2. All three reviewers raise important questions about some of the immunoblot data presented in the paper. Specifically, Reviewer 1 points out that the use of PSTAIR as a loading control requires some clarification. Furthermore, Reviewers 2 and 3 point out that some of the blots shown (e.g., Figure 1B and Figure 5) should include quantitation.

3. Both Reviewer 2 and Reviewer 3 commented on the somewhat unexpected phenotype of the GFA1-3A mutant. Although the reviewers agree that the genetic tests shown in Figure 4F support the authors’ interpretations, it is still unclear why the GFA1-3A and the isr1 deletion exhibit similar phenotypes. Perhaps this point warrants additional discussion.

Reviewer 3 also points out that the data in this paper do not provide evidence that Isr1 directly phosphorylates Gfa1. While such evidence would strengthen the assertions of the manuscript, there is consensus that this should not be a criteria for publication. Additionally, there are several other comments made by reviewers that the authors should attempt to address with minor revisions to the text of the manuscript.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Please be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Jason MacGurn

Guest Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: Manuscript: PGENETICS-D-20-00243

TITLE: The kinase Isr1 negatively regulates hexosamine biosynthesis in S. cerevisiae

AUTHOR(S): David P Toczyski

Summary. Yeast encode 117 protein kinases in the superfamily of eukaryotic protein kinases, plus a smattering of atypical protein kinases related in function but not evolutionary lineage. Almost all of the yeast kinases have been characterized at least to some extent. This paper determines the function of one of the few remaining poorly characterized protein kinases in yeast. The experiments presented here show the following 1) they demonstrate the role of ISR1 in regulating the hexosamine pathway; 2) they use phosphoproteomic data to identify GFA1 as the ISR1 substrate and 3) they show the regulation of ISR1 abundance via a PHO85-targeted phosphodegron. These results are significant and novel. A few changes to the manuscript that could strengthen the paper are noted below.

1. ISR1 and Raf/Ras signaling. The first paper to describe ISR1 is the Miyahara paper from 1998. This paper states that ISR1 “has the highest degree of similarity to the Raf proto-oncogene from mouse”. This statement is demonstrably false. Unfortunately, the only other paper on ISR1 (Mehlgarten 2007) repeats this claim, adding credibility to it. This manuscript should break this chain of errors and not repeat it anymore. ISR1 is NOT related to Raf. Mammalian Raf kinases are members of the tyrosine kinase-like subfamily (Manning 2002). Yeast does not contain any members of this family. ISR1 is not the orthologue of Raf. Of the 117 kinases in yeast, 79 show greater homology to the kinase domain of mouse Raf than ISR1. The 30% identity reported in the Miyahara paper is extremely misleading since that homology is limited to a 109 residue peptide and not the entire kinase domain. The misinformation about a Raf-ISR1 connection needs to stop now. Further, the introduction should delete the whole paragraph on Raf kinase and Ras signaling. A more accurate and factually correct description of ISR1 and its place in the kinomes of yeast and humans would be greatly appreciated. ISR1 is an unusual kinase and has variant sequences in the place of what are usually invariant kinase signature motifs. For instance, the catalytic aspartate that these authors mutate (D280) is usually in the “HRD” motif. In ISR1, this sequence is HGD. Because of its unusual sequence, many databases list ISR1 as a “putative protein kinase”. That is much more accurate and pertinent to this paper than any discussion of Raf.

The connection of ISR1 to Raf and Ras signaling reappears in the discussion where the authors note that the closest relative of ISR1 in S. pombe is the kinase byr2. This is true, but the similarity is very weak and not reciprocal. S. pombe byr2 is more likely to be the orthologue of S. cerevisiae Ste11. If you BLAST pombe byr2 against the S. cerevisiae proteome, the top hit is STE11, not ISR1. In fact, 103 of the 117 yeast kinases show stronger homology to pombe byr2 than does ISR1. S. pombe does not have a clear orthologue of ISR1; there is no connection to Raf or RAS. This paragraph in the discussion should be removed.

2. Staurosporine. ISR1 will be forever linked to staurosporine since ISR stands for “Inhibition of Staurosporine Resistance”. Miyahara et al. in 1998 describe staurosporine as “a potent inhibitor of protein kinase C”. This may be true but it is not the whole story. We now know that staurosporine is a potent inhibitor of almost all protein kinases. It binds to the ATP binding pocket with higher affinity than ATP. In fact, its presence in the ATP pocket stabilizes kinase domain structures explaining why this compound is included during crystallization of so many protein kinases. Probably the most relevant data in the Miyahara paper is that isr1 mutants exacerbate a pkc1 mutant, thus connecting both kinases to the cell wall integrity pathway. Perhaps when referring to staurosporine as a PKC inhibitor, they could add that this compound is really a broad spectrum kinase inhibitor.

3. PSTAIR. Many western blot experiments in this paper use PSTAIR antibody as a loading control (Figs 1,5 and 6). This needs to be explained. My understanding is that PSTAIR is a monoclonal that recognizes the PSTAIR sequence in cdk kinases. Yeast has 2 cdk’s with the PSTAIR sequence, Cdc28 and Pho85. These two proteins have similar molecular weights: 34.0 vs 34.9 kDa. What is being recognized in the PSTAIR blots? What is the MW range being shown in the PSTAIR panels? This may be an odd control blot to choose since Pho85 becomes an important player in the control of ISR1 abundance. There is no mention of the PSTAIR panel in the figure legends.

4. ISR1 deletion details. The authors note that the isr1∆:KAN allele in the MATa collection has RNA processing phenotypes caused by interference with the adjacent gene. They make a new isr1∆ strain with a different marker. It would be worth including the details of how their new deletion allele differs from the KAN allele. How did they make an isr1 deletion that doesn’t interfere with the adjacent gene?

5. Improving the discussion. I have noted above some topics that should be removed from this paper. The one topic that should be added is a better discussion of how their new data relates to the data in the Mehlgarten paper. That paper is the best description of ISR1 to date and connects ISR1 to the cell wall and PKC1. It’s a bit unfair to the Mehlgarten authors to call ISR1 a “previously uncharacterized kinase” (line 418). Poorly characterized, yes; uncharacterized, no.

Reviewer #2: Review PloS Genetics

Although protein glycosylation is an essential and well-studied cellular process, how protein glycosylation might be regulated throughout the cell cycle and in response to stress is not clear. Here, this report focuses on a protein kinase and negative regulator of an early step in protein glycosylation, which is the biosynthesis of an N-linked sugar precursor. This negative regulatory kinase is regulated by the alternate CDK Pho85, which allows cells to alter the levels of the regulator according to the cell’s nutrient needs.

There are many strengths to this study. It is very well written. The data is clear and carefully interpreted. The findings are impactful and presented in a clear and logical manner. The combination of phenotypic and genetic data with immunoblot and MASS SPEC data, plus previous data from the lab and the community lends credence to the study.

The main piece of data that is missing is direct demonstration that Isr1 phosphorylates Gfa1. Typically, this can be shown in two ways, a shift in protein levels by immunoblot (e.g. phos-tag) analysis (which itself is indirect) and by in vitro kinase assays. The authors do present MASS SPEC data to address this point.

Below are some minor comments.

1. Are there homologs to Isr1?

2. Immunoblots can be quantitated. By adjusting levels to the loading control, setting it to 1 and comparing other levels to that (Fig. 1B). Numbers can be represented below the IBs.

3. What is the significance of the fact that the levels of overexpressed ISR1 are the same as Pkc1 in the cell?

4. Section heading and within the paragraph “Isr1 overexpression is synthetic lethal with enzymes in the hexosamine biosynthesis pathway’ should be synthetically

5. Does the fact that Gfa1 -3A cells are sensitive to tunacamycin imply that phosphorylation of Gfa1 is important for balancing the cell’s protein glycosylation needs?

6. Given that Isr1 is cell cycle regulated, it might be phosphorylated by the cell’s major CDK, Cdc28?

7. Plate labels should appear above not below the pictures.

8. In Figure 5, it looks like cdc4-1 is much more important than cdc53-1 in stablilzation of Isr1 levels. Again, quantitation of protein levels might help this analysis.

9. wildtype should be wild-type

10. What do the authors think about all the other proteins that are being phosphorylated by Isr1?

11. Are the glyosylation profiles of select glycosylated proteins impacted by these mutants, observed for example by IB analysis?

Reviewer #3: This is a well-written and compelling story that reveals a function for the yeast Raf homolog, Isr1. Deletion of ISR1 confers tolerance to N-glycosylation inhibitor tunicamycin, whereas its moderate overexpression confers tolerance to the chitin antagonist calcofluor white. Based on these phenotypes, the authors pursued the possibility that ISR1 is a negative regulator of hexosamine biosynthesis, specifically UDP-N-acetyl glucosamine (UDP-GlcNAc) production by GFA1. UDP-GlcNAc is required for the first steps in N-glycosylation and GPI-anchor biosynthesis, as well as for the production of chitin. The authors showed that strong ISR1 overexpression causes growth inhibition, which was suppressed either by over-expression of GFA1, or by addition of glucosamine to the medium, which is scavenged to produce UDP-GlcNAc independently of GFA1.

The authors go on to show that Gfa1 is phosphorylated on neighboring residues in an Isr1-dependent manner, mutation of which to Ala (GFA1-3A) overcame the growth inhibition associated with overexpression of ISR1, supporting the conclusion that Isr1 phosphorylation of Gfa1 is inhibitory. One result that was not entirely consistent with this conclusion was the finding that the GFA1-3A mutation did not confer tolerance to tunicamycin (Fig. 4E), as would be expected if it was to phenocopy deletion of ISR1. The authors explain this with a demonstration that the GFA1-3A mutation is somewhat hypomorphic and causes tunicamycin sensitivity by itself. They further suggest that Isr1 has other targets that contribute to its role in N-glycosylation. This is a reasonable conjecture.

Finally, the authors show that the N-terminal regulatory domain of Isr1 contains a phosphodegron that is phosphorylated by Pho85 and targeted by the SCF-Cdc4 ubiquitylation complex. This confers tight cell cycle regulation of Isr1 levels so that its activity is largely confined to the G1/S transition. These experiments set out in Fig. 5, should be accompanied by quantitation of the immunoblot bands. Aside from this lone criticism, this manuscript is viewed to be very strong.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

PLoS Genet. 2020 Jun 24;16(6):e1008840. doi: 10.1371/journal.pgen.1008840.r002

Author response to Decision Letter 0

23 Apr 2020

Attachment

Submitted filename: Plos Genetics response to reviewers .pdf

Click here for additional data file.^{(141.4KB, pdf)}

PLoS Genet. doi: 10.1371/journal.pgen.1008840.r003

Decision Letter 1

Gregory P Copenhaver, Jason MacGurn

8 May 2020

Dear Dr Toczyski,

We are pleased to inform you that your manuscript entitled "The kinase Isr1 negatively regulates hexosamine biosynthesis in S. cerevisiae" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional accept, but your manuscript will not be scheduled for publication until the required changes have been made.

Once your paper is formally accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plosgenetics@plos.org.

In the meantime, please log into Editorial Manager at https://www.editorialmanager.com/pgenetics/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production and billing process. Note that PLOS requires an ORCID iD for all corresponding authors. Therefore, please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

If you have a press-related query, or would like to know about one way to make your underlying data available (as you will be aware, this is required for publication), please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Jason MacGurn

Guest Editor

PLOS Genetics

Gregory P. Copenhaver

Editor-in-Chief

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

----------------------------------------------------

Data Deposition

If you have submitted a Research Article or Front Matter that has associated data that are not suitable for deposition in a subject-specific public repository (such as GenBank or ArrayExpress), one way to make that data available is to deposit it in the Dryad Digital Repository. As you may recall, we ask all authors to agree to make data available; this is one way to achieve that. A full list of recommended repositories can be found on our website.

The following link will take you to the Dryad record for your article, so you won't have to re‐enter its bibliographic information, and can upload your files directly:

http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-20-00243R1

More information about depositing data in Dryad is available at http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.

Additionally, please be aware that our data availability policy requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.

----------------------------------------------------

Press Queries

If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via plosgenetics@plos.org.

PLoS Genet. doi: 10.1371/journal.pgen.1008840.r004

Acceptance letter

Gregory P Copenhaver, Jason MacGurn

18 Jun 2020

PGENETICS-D-20-00243R1

The kinase Isr1 negatively regulates hexosamine biosynthesis in <I>S. cerevisiae</I>

Dear Dr Toczyski,

We are pleased to inform you that your manuscript entitled "The kinase Isr1 negatively regulates hexosamine biosynthesis in <I>S. cerevisiae</I>" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

Soon after your final files are uploaded, unless you have opted out or your manuscript is a front-matter piece, the early version of your manuscript will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work!

With kind regards,

Kaitlin Butler

PLOS Genetics

On behalf of:

The PLOS Genetics Team

Carlyle House, Carlyle Road, Cambridge CB4 3DN | United Kingdom

plosgenetics@plos.org | +44 (0) 1223-442823

plosgenetics.org | Twitter: @PLOSGenetics

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. ISR1 lethality is not rescued by osmotic stabilizers or other carbon sources.

(TIF)

Click here for additional data file.^{(2.5MB, tif)}

S2 Fig. ISR1 does not affect Gfa1 protein levels.

(TIF)

Click here for additional data file.^{(3.1MB, tif)}

S3 Fig. Isr1 is partially stabilized in non-fermentable carbon sources.

(TIF)

Click here for additional data file.^{(478KB, tif)}

S4 Fig. Mutation of an Isr1 phosphodegron confers resistance to Congo Red.

(A) An Isr1 phosphodegron mutant is resistant to Congo Red. Strains of the indicated genotypes were diluted onto YPD in the presence or absence of 150 μg/ml Congo Red.

(TIF)

Click here for additional data file.^{(696.2KB, tif)}

S5 Fig. MAPKs and PRR1 are not downstream of ISR1.

(TIF)

Click here for additional data file.^{(2MB, tif)}

S6 Fig. ISR1 phosphodegron mutant sequence.

Double-stranded DNA sequence used to generate ISR1 phosphodegron mutant. Blue sequence is the end of the ISR1 promoter. Lowercase red base pairs indicate mutated residues.

(DOCX)

Click here for additional data file.^{(112.7KB, docx)}

S1 Table. Phosphoproteomics dataset for isr1Δ vs GAL-2μ ISR1.

(XLSX)

Click here for additional data file.^{(743.3KB, xlsx)}

S2 Table. Strains used in this study.

(DOCX)

Click here for additional data file.^{(120.9KB, docx)}

S3 Table. Plasmids used in this study.

(DOCX)

Click here for additional data file.^{(84.1KB, docx)}

Attachment

Submitted filename: Plos Genetics response to reviewers .pdf

Click here for additional data file.^{(141.4KB, pdf)}

Data Availability Statement

[pgen.1008840.ref001] 1.Ptacek J, Devgan G, Michaud G, Zhu H, Zhu X, Fasolo J, et al. Global analysis of protein phosphorylation in yeast. Nature. 2005;438: 679–684. 10.1038/nature04187 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref002] 2.Manning G, Plowman GD, Hunter T, Sudarsanam S. Evolution of protein kinase signaling from yeast to man. Trends Biochem Sci. 2002;27: 514–520. 10.1016/s0968-0004(02)02179-5 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref003] 3.Fiedler D, Braberg H, Mehta M, Chechik G, Cagney G, Mukherjee P, et al. Functional Organization of the S. cerevisiae Phosphorylation Network. Cell. 2009;136: 952–963. 10.1016/j.cell.2008.12.039 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref004] 4.Sharma K, D’Souza RCJ, Tyanova S, Schaab C, Wiśniewski JR, Cox J, et al. Ultradeep Human Phosphoproteome Reveals a Distinct Regulatory Nature of Tyr and Ser/Thr-Based Signaling. Cell Rep. 2014;8: 1583–1594. 10.1016/j.celrep.2014.07.036 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref005] 5.Kannan N, Taylor SS, Zhai Y, Venter JC, Manning G. Structural and Functional Diversity of the Microbial Kinome. PLOS Biol. 2007;5: e17 10.1371/journal.pbio.0050017 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref006] 6.Miyahara K, Hirata D, Miyakawa T. Functional Interaction of Isr1, a Predicted Protein Kinase, with the Pkc1 Pathway in Saccharomyces cerevisiae. Biosci Biotechnol Biochem. 1998;62: 1376–1380. 10.1271/bbb.62.1376 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref007] 7.Levin DE. Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway. Genetics. 2011;189: 1145–1175. 10.1534/genetics.111.128264 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref008] 8.Mehlgarten C, Zink S, Rutter J, Schaffrath R. Dosage suppression of the Kluyveromyces lactis zymocin by Saccharomyces cerevisiae ISR1 and UGP1. FEMS Yeast Res. 2007;7: 722–730. 10.1111/j.1567-1364.2007.00216.x [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref009] 9.Jablonowski D, Fichtner L, Martin VJ, Klassen R, Meinhardt F, Stark MJR, et al. Saccharomyces cerevisiae cell wall chitin, the Kluyveromyces lactis zymocin receptor. Yeast. 2001;18: 1285–1299. 10.1002/yea.776 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref010] 10.Orlean P. Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall. Genetics. 2012;192: 775–818. 10.1534/genetics.112.144485 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref011] 11.Larkin A, Imperiali B. The Expanding Horizons of Asparagine-Linked Glycosylation. Biochemistry. 2011;50: 4411–4426. 10.1021/bi200346n [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref012] 12.Schekman R, Brawley V. Localized deposition of chitin on the yeast cell surface in response to mating pheromone. Proc Natl Acad Sci U S A. 1979;76: 645–649. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC383005/ [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref013] 13.Lagorce A, Berre-Anton VL, Aguilar-Uscanga B, Martin-Yken H, Dagkessamanskaia A, François J. Involvement of GFA1, which encodes glutamine–fructose-6-phosphate amidotransferase, in the activation of the chitin synthesis pathway in response to cell-wall defects in Saccharomyces cerevisiae. Eur J Biochem. 2002;269: 1697–1707. 10.1046/j.1432-1327.2002.02814.x [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref014] 14.Lesage G, Bussey H. Cell Wall Assembly in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2006;70: 317–343. 10.1128/MMBR.00038-05 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref015] 15.Bulik DA, Olczak M, Lucero HA, Osmond BC, Robbins PW, Specht CA. Chitin Synthesis in Saccharomyces cerevisiae in Response to Supplementation of Growth Medium with Glucosamine and Cell Wall Stress. Eukaryot Cell. 2003;2: 886–900. 10.1128/ec.2.5.886-900.2003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref016] 16.Valdivia RH, Schekman R. The yeasts Rho1p and Pkc1p regulate the transport of chitin synthase III (Chs3p) from internal stores to the plasma membrane. Proc Natl Acad Sci. 2003;100: 10287 10.1073/pnas.1834246100 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref017] 17.Milewski S. Glucosamine-6-phosphate synthase—the multi-facets enzyme. Biochim Biophys Acta BBA—Protein Struct Mol Enzymol. 2002;1597: 173–192. 10.1016/S0167-4838(02)00318-7 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref018] 18.Milewski S, Gabriel I, Olchowy J. Enzymes of UDP-GlcNAc biosynthesis in yeast. Yeast. 2006;23: 1–14. 10.1002/yea.1337 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref019] 19.Zibrova D, Vandermoere F, Göransson O, Peggie M, Mariño KV, Knierim A, et al. GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis. Biochem J. 2017;474: 983–1001. 10.1042/BCJ20160980 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref020] 20.Graack HR, Cinque U, Kress H. Functional regulation of glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1) of Drosophila melanogaster in a UDP-N-acetylglucosamine and cAMP-dependent manner. Biochem J. 2001;360: 401–412. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1222241/ [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref021] 21.Zheng J, Khalil M, Cannon JF. Glc7p Protein Phosphatase Inhibits Expression of Glutamine-Fructose-6-phosphate Transaminase from GFA1. J Biol Chem. 2000;275: 18070–18078. 10.1074/jbc.M000918200 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref022] 22.Milewski S, Kuszczak D, Jedrzejczak R, Smith RJ, Brown AJP, Gooday GW. Oligomeric Structure and Regulation of Candida albicans Glucosamine-6-phosphate Synthase. J Biol Chem. 1999;274: 4000–4008. 10.1074/jbc.274.7.4000 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref023] 23.Chang Q, Su K, Baker JR, Yang X, Paterson AJ, Kudlow JE. Phosphorylation of Human Glutamine:Fructose-6-phosphate Amidotransferase by cAMP-dependent Protein Kinase at Serine 205 Blocks the Enzyme Activity. J Biol Chem. 2000;275: 21981–21987. 10.1074/jbc.M001049200 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref024] 24.Ghaemmaghami S, Huh W-K, Bower K, Howson RW, Belle A, Dephoure N, et al. Global analysis of protein expression in yeast. Nature. 2003;425: 737–741. 10.1038/nature02046 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref025] 25.Giaever G, Chu AM, Ni L, Connelly C, Riles L, Véronneau S, et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature. 2002;418: 387–391. 10.1038/nature00935 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref026] 26.Holbein S, Wengi A, Decourty L, Freimoser FM, Jacquier A, Dichtl B. Cordycepin interferes with 3’ end formation in yeast independently of its potential to terminate RNA chain elongation. RNA. 2009;15: 837–849. 10.1261/rna.1458909 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref027] 27.Yoo J, Mashalidis EH, Kuk ACY, Yamamoto K, Kaeser B, Ichikawa S, et al. GlcNAc-1-P-transferase-tunicamycin complex structure reveals basis for inhibition of N-glycosylation. Nat Struct Mol Biol. 2018;25: 217–224. 10.1038/s41594-018-0031-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref028] 28.Ben-Shitrit T, Yosef N, Shemesh K, Sharan R, Ruppin E, Kupiec M. Systematic identification of gene annotation errors in the widely used yeast mutation collections. Nat Methods. 2012;9: 373–378. 10.1038/nmeth.1890 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref029] 29.Barnes G, Hansen WJ, Holcomb CL, Rine J. Asparagine-linked glycosylation in Saccharomyces cerevisiae: genetic analysis of an early step. Mol Cell Biol. 1984;4: 2381–2388. 10.1128/mcb.4.11.2381 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref030] 30.Ram AFJ, Klis FM. Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red. Nat Protoc. 2006;1: 2253–2256. 10.1038/nprot.2006.397 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref031] 31.Ram AFJ, Wolters A, Hoopen RT, Klis FM. A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast. 1994;10: 1019–1030. 10.1002/yea.320100804 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref032] 32.Roncero C, Valdivieso MH, Ribas JC, Durán A. Isolation and characterization of Saccharomyces cerevisiae mutants resistant to Calcofluor white. J Bacteriol. 1988;170: 1950–1954. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC211056/ [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref033] 33.Sobering AK, Watanabe R, Romeo MJ, Yan BC, Specht CA, Orlean P, et al. Yeast Ras Regulates the Complex that Catalyzes the First Step in GPI-Anchor Biosynthesis at the ER. Cell. 2004;117: 637–648. 10.1016/j.cell.2004.05.003 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref034] 34.Mitchell AP. The GLN1 Locus of SACCHAROMYCES CEREVISIAE Encodes Glutamine Synthetase. Genetics. 1985;111: 243–258. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1202641/ [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref035] 35.Watzele G, Tanner W. Cloning of the glutamine:fructose-6-phosphate amidotransferase gene from yeast. Pheromonal regulation of its transcription. J Biol Chem. 1989;264: 8753–8758. Available: http://www.jbc.org/content/264/15/8753 [PubMed] [Google Scholar]

[pgen.1008840.ref036] 36.Gomez A, Perez J, Reyes A, Duran A, Roncero C. Slt2 and Rim101 Contribute Independently to the Correct Assembly of the Chitin Ring at the Budding Yeast Neck in Saccharomyces cerevisiae. Eukaryot Cell. 2009;8: 1449–1459. 10.1128/EC.00153-09 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref037] 37.Eguchi S, Oshiro N, Miyamoto T, Yoshino K, Okamoto S, Ono T, et al. AMP-activated protein kinase phosphorylates glutamine : fructose-6-phosphate amidotransferase 1 at Ser243 to modulate its enzymatic activity. Genes Cells. 2009;14: 179–189. 10.1111/j.1365-2443.2008.01260.x [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref038] 38.Frisa PS, Sonneborn DR. Developmentally regulated interconversions between end product-inhibitable and noninhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by protein dephosphorylation-phosphorylation reactions. Proc Natl Acad Sci. 1982;79: 6289–6293. 10.1073/pnas.79.20.6289 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref039] 39.Breitkreutz A, Choi H, Sharom JR, Boucher L, Neduva V, Larsen B, et al. A Global Protein Kinase and Phosphatase Interaction Network in Yeast. Science. 2010;328: 1043–1046. 10.1126/science.1176495 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref040] 40.Holt LJ, Tuch BB, Villén J, Johnson AD, Gygi SP, Morgan DO. Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution. Science. 2009;325: 1682 10.1126/science.1172867 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref041] 41.Spellman PT, Sherlock G, Zhang MQ, Iyer VR, Anders K, Eisen MB, et al. Comprehensive Identification of Cell Cycle–regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray Hybridization. Mol Biol Cell. 1998;9: 3273–3297. 10.1091/mbc.9.12.3273 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref042] 42.Mark KG, Simonetta M, Maiolica A, Seller CA, Toczyski DP. Ubiquitin Ligase Trapping Identifies an SCFSaf1 Pathway Targeting Unprocessed Vacuolar/Lysosomal Proteins. Mol Cell. 2014;53: 148–161. 10.1016/j.molcel.2013.12.003 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref043] 43.Willems AR, Schwab M, Tyers M. A hitchhiker’s guide to the cullin ubiquitin ligases: SCF and its kin. Biochim Biophys Acta BBA—Mol Cell Res. 2004;1695: 133–170. 10.1016/j.bbamcr.2004.09.027 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref044] 44.Tang X, Orlicky S, Liu Q, Willems A, Sicheri F, Tyers M. Genome-Wide Surveys for Phosphorylation-Dependent Substrates of SCF Ubiquitin Ligases. Methods in Enzymology. Academic Press; 2005. pp. 433–458. 10.1016/S0076-6879(05)99030-7 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref045] 45.Skowyra D, Craig KL, Tyers M, Elledge SJ, Harper JW. F-Box Proteins Are Receptors that Recruit Phosphorylated Substrates to the SCF Ubiquitin-Ligase Complex. Cell. 1997;91: 209–219. 10.1016/s0092-8674(00)80403-1 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref046] 46.Hao B, Oehlmann S, Sowa ME, Harper JW, Pavletich NP. Structure of a Fbw7-Skp1-Cyclin E Complex: Multisite-Phosphorylated Substrate Recognition by SCF Ubiquitin Ligases. Mol Cell. 2007;26: 131–143. 10.1016/j.molcel.2007.02.022 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref047] 47.Lyons NA, Fonslow BR, Diedrich JK, Yates JR, Morgan DO. Sequential Primed Kinases Create a Damage-Responsive Phosphodegron on Eco1. Nat Struct Mol Biol. 2013;20: 194–201. 10.1038/nsmb.2478 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref048] 48.Dephoure N, Howson RW, Blethrow JD, Shokat KM, O’Shea EK. Combining chemical genetics and proteomics to identify protein kinase substrates. Proc Natl Acad Sci. 2005;102: 17940–17945. 10.1073/pnas.0509080102 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref049] 49.Carroll AS, Bishop AC, DeRisi JL, Shokat KM, O’Shea EK. Chemical inhibition of the Pho85 cyclin-dependent kinase reveals a role in the environmental stress response. Proc Natl Acad Sci U S A. 2001;98: 12578–12583. 10.1073/pnas.211195798 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref050] 50.Huang D, Friesen H, Andrews B. Pho85, a multifunctional cyclin-dependent protein kinase in budding yeast. Mol Microbiol. 2007;66: 303–314. 10.1111/j.1365-2958.2007.05914.x [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref051] 51.Espinoza FH, Ogas J, Herskowitz I, Morgan DO. Cell cycle control by a complex of the cyclin HCS26 (PCL1) and the kinase PHO85. Science. 1994;266: 1388–1391. 10.1126/science.7973730 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref052] 52.Schneider KR, Smith RL, O’Shea EK. Phosphate-regulated inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81. Science. 1994;266: 122–126. 10.1126/science.7939631 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref053] 53.Huang D, Moffat J, Wilson WA, Moore L, Cheng C, Roach PJ, et al. Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10. Mol Cell Biol. 1998;18: 3289–3299. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC108910/ [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref054] 54.Imai K, Noda Y, Adachi H, Yoda K. A Novel Endoplasmic Reticulum Membrane Protein Rcr1 Regulates Chitin Deposition in the Cell Wall of Saccharomyces cerevisiae. J Biol Chem. 2005;280: 8275–8284. 10.1074/jbc.M409428200 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref055] 55.Sopko R, Huang D, Preston N, Chua G, Papp B, Kafadar K, et al. Mapping Pathways and Phenotypes by Systematic Gene Overexpression. Mol Cell. 2006;21: 319–330. 10.1016/j.molcel.2005.12.011 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref056] 56.Levin DE. Cell Wall Integrity Signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2005;69: 262–291. 10.1128/MMBR.69.2.262-291.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref057] 57.Bandyopadhyay S, Mehta M, Kuo D, Sung M-K, Chuang R, Jaehnig EJ, et al. Rewiring of Genetic Networks in Response to DNA Damage. Science. 2010;330: 1385–1389. 10.1126/science.1195618 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref058] 58.Costanzo M, VanderSluis B, Koch EN, Baryshnikova A, Pons C, Tan G, et al. A global genetic interaction network maps a wiring diagram of cellular function. Science. 2016;353: aaf1420 10.1126/science.aaf1420 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref059] 59.Perez-Riverol Y, Csordas A, Bai J, Bernal-Llinares M, Hewapathirana S, Kundu DJ, et al. The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 2019;47: D442–D450. 10.1093/nar/gky1106 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref060] 60.Meier F, Brunner A-D, Koch S, Koch H, Lubeck M, Krause M, et al. Online Parallel Accumulation–Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer. Mol Cell Proteomics MCP. 2018;17: 2534–2545. 10.1074/mcp.TIR118.000900 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pgen.1008840.ref061] 61.Meier F, Brunner A-D, Frank M, Ha A, Voytik E, Kaspar-Schoenefeld S, et al. Parallel accumulation–serial fragmentation combined with data-independent acquisition (diaPASEF): Bottom-up proteomics with near optimal ion usage. bioRxiv. 2019; 656207 10.1101/656207 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref062] 62.Choi M, Chang C-Y, Clough T, Broudy D, Killeen T, MacLean B, et al. MSstats: an R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments. Bioinforma Oxf Engl. 2014;30: 2524–2526. 10.1093/bioinformatics/btu305 [DOI] [PubMed] [Google Scholar]

[pgen.1008840.ref063] 63.Nolte H, MacVicar TD, Tellkamp F, Krüger M. Instant Clue: A Software Suite for Interactive Data Visualization and Analysis. Sci Rep. 2018;8 10.1038/s41598-018-31154-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

The kinase Isr1 negatively regulates hexosamine biosynthesis in S. cerevisiae

Emma B Alme

Erica Stevenson

Nevan J Krogan

Danielle L Swaney

David P Toczyski

Roles

Abstract

Author summary

Introduction

Results

Overexpression of Isr1 kinase activity is lethal

Fig 1. Overexpression of ISR1 is lethal.

ISR1 does not function in RNA processing

Fig 2. Altering ISR1 dosage confers phenotypes associated with changes in flux through the hexosamine biosynthesis pathway.

ISR1 overexpression has phenotypes associated with a deficiency in UDP-GlcNAc

ISR1 overexpression is synthetically lethal with enzymes in the hexosamine biosynthesis pathway

ISR1 lethality is rescued by exogenous glucosamine, but not other precursors to UDP-GlcNAc synthesis

Fig 3. Exogenous glucosamine specifically rescues ISR1 overexpression phenotypes.

Isr1 is a negative regulator of Gfa1

Fig 4. Gfa1 is phosphorylated in an Isr1-dependent manner and a GFA1 phosphomutant rescues ISR1 lethality.

Isr1 is an unstable cell-cycle regulated protein targeted by Pho85 and Cdc4

Fig 5. Isr1 is an unstable protein targeted by Pho85 and Cdc4.

Stabilization of an endogenous Isr1 phosphodegron recapitulates Isr1 overexpression phenotypes

Fig 6. Stabilization of an endogenous Isr1 phosphodegron recapitulates overexpression phenotypes.

Discussion

Materials and methods

Strains and plasmids

Yeast cell culture

Western blotting

Cell cycle arrest

Cycloheximide-chase assays

Mass spectrometry sample preparation

Phosphopeptide enrichment by immobilized metal affinity chromatography

Proteomic data acquisition and analysis

Spot tests

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Gregory P Copenhaver

Jason MacGurn

Roles

Author response to Decision Letter 0

Decision Letter 1

Gregory P Copenhaver

Jason MacGurn

Roles

Acceptance letter

Gregory P Copenhaver

Jason MacGurn

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases