The yeast protein Gdt1p transports Mn²⁺ ions and thereby regulates manganese homeostasis in the Golgi

Abstract

The uncharacterized protein family 0016 (UPF0016) is a family of secondary ion transporters implicated in calcium homeostasis and some diseases. More precisely, genetic variants of the human UPF0016 ortholog transmembrane protein 165 (TMEM165) have been linked to congenital disorders of glycosylation (CDG). The Saccharomyces cerevisiae ortholog Gdt1p has been shown to be involved in calcium homeostasis and protein glycosylation. Moreover, plant and bacterial UPF0016 members appear to have putative roles in Mn²⁺ homeostasis. Here, we produced the yeast UPF0016 member Gdt1p in the bacterial host Lactococcus lactis. Using Mn²⁺-induced quenching of Fura-2–emitted fluorescence, we observed that Gdt1p mediates Mn²⁺ influx, in addition to its previously reported regulation of Ca²⁺ influx. The estimated K_m values of Gdt1p of 15.6 ± 2.6 μm for Ca²⁺ and 83.2 ± 9.8 μm for Mn²⁺ indicated that Gdt1p has a higher affinity for Ca²⁺ than for Mn²⁺. In yeast cells, we found that Gdt1p is involved in the resistance to high Mn²⁺ concentration and controls total Mn²⁺ stores. Lastly, we demonstrated that GDT1 deletion affects the activity of the yeast Mn²⁺-dependent Sod2p superoxide dismutase, most likely by modulating cytosolic Mn²⁺ concentrations. Taken together, we obtained first evidence that Gdt1p from yeast directly transports manganese, which strongly reinforces the suggested link between the UPF0016 family and Mn²⁺ homeostasis and provides new insights into the molecular causes of human TMEM165-associated CDGs. Our results also shed light on how yeast cells may regulate Golgi intraluminal concentrations of manganese, a key cofactor of many enzymes involved in protein glycosylation.

Introduction

The uncharacterized protein family 0016 (UPF0016)³ gathers highly conserved membrane proteins that are widely distributed among kingdoms and defined by the presence of one or two copies of the EXGD(K/R)(T/S) motif (1). It was previously reported that specific mutations in the human UPF0016 member transmembrane protein 165 (TMEM165) cause congenital disorders of glycosylation (2). Because the exact function of the UPF0016 members is not deciphered yet, the causal link between a mutated TMEM165 and congenital disorders of glycosylation remains unclear. Based on previous studies, the UPF0016 members were hypothesized to act as Ca²⁺/H⁺ antiporters. This hypothesis arose, among others, from studies carried out on the Saccharomyces cerevisiae UPF0016 member Gdt1p. Indeed, we previously reported that Gdt1p localizes at the yeast Golgi membrane and that this protein is required for (i) Ca²⁺ tolerance, (ii) proper Ca²⁺ response after exposure to salt stress, and (iii) proper proteins glycosylation in the presence of a high calcium concentration (3, 4). Transport of Ca²⁺ by Gdt1p was further demonstrated by producing Gdt1p in the bacterial host Lactococcus lactis and using the fluorescent Ca²⁺-sensitive Fura-2 probe (4). The acidic and polar uncharged amino acids of the conserved motif EXGD(K/R)(T/S) found in Gdt1p were additionally demonstrated to be essential for proper Ca²⁺ tolerance and Ca²⁺ response to salt stress (5).

In addition to this role in calcium homeostasis, recent studies on other UPF0016 members suggested an implication of these proteins in manganese homeostasis. Among others, Wang et al. (6) and Schneider et al. (7) reported an implication of the Arabidopsis thaliana CCHA1 in resistance to high concentrations of Ca²⁺ and Mn²⁺ and to pH changes. The UPF0016 members of the bacterium Vibrio cholerae and of the cyanobacterial model strain Synechocystis sp. PCC 6803 were additionally suggested to function as Mn²⁺ transporters (8–10). Interestingly, members of the photosynthetic A. thaliana and Synechocystis both localize to the thylakoid membrane and are essential for proper photosynthesis, most likely because of the required presence of Mn²⁺ in the oxygen-evolving complex of photosystem II (6–8, 10). Regarding the yeast Gdt1p, we previously reported that the glycosylation defects observed in a gdt1Δ strain grown in the presence of a high calcium concentration were suppressed when manganese was additionally present in the culture medium (4, 11). Manganese cations were also demonstrated to restore glycosylation in TMEM165-deficient cells (11). Interestingly, TMEM165 and a Myc-tagged version of Gdt1p were both reported to be degraded in the presence of high extracellular manganese concentration (12, 13). However, although these studies all established a clear functional link between the UPF0016 members and manganese homeostasis, none of them provided direct evidence for Mn²⁺ transport.

In yeast, the mechanisms by which cells regulate their Ca²⁺ and Mn²⁺ homeostasis are not fully characterized yet. This statement also applies to the Golgi where a proper maintenance of the homeostasis of these two ions is known to be essential for numerous cellular processes including glycosylation. Calcium, well described to regulate the intravesicular trafficking between the different organelles, indirectly contributes to glycosylation because the transport of proteins along the secretory pathway is affected by calcium concentration (14). Ca²⁺ is additionally described as essential for the activity and stability of enzymes involved in glycosylation (15, 16). On the other hand, the mannosyltransferases Och1p, Mnn9p, Mnn1p, Mnn2p, and Mnn5p, all implicated in N-glycosylation, require Mn²⁺ as cofactor (17–20). A tight regulation of both Ca²⁺ and Mn²⁺ concentrations in the Golgi is therefore essential. By now, the P-type ATPase Pmr1p is the only reported yeast Golgi transporter of Ca²⁺ and Mn²⁺. By analyzing the contribution of Pmr1p and Gdt1p in the Golgi glycosylation process, Dulary et al. (13) suggested that the abundance and function of Gdt1p depends on the function of Pmr1p, thereby creating a functional link between these two Golgi proteins. Our study highly reinforces the hypothesis that Gdt1p and Pmr1p act in concert in the regulation of the yeast Ca²⁺ and Mn²⁺ homeostasis.

In this paper, we combined approaches in bacteria as a system to carry out direct transport assays and in yeast for a better understanding of the physiological role of Gdt1p. First, in bacteria, we (i) directly showed transport of Mn²⁺ by Gdt1p, (ii) highlighted a competition between manganese and calcium for transport by Gdt1p, and (iii) estimated a higher affinity of the transporter for calcium than for manganese. Second, in yeast, we (i) demonstrated that Gdt1p is involved in resistance to high Mn²⁺ concentration and controls the total Mn²⁺ stores and (ii) showed that the activity of the Mn²⁺-dependent Sod2p is modulated by a deletion of GDT1. Altogether, this study undeniably demonstrates that Gdt1p and most likely other UPF0016 members involved in essential cellular processes like glycosylation or photosynthesis are directly involved in Mn²⁺ transport. Additionally, this study places Gdt1p as a novel yeast Golgi protein which, together with Pmr1p, tightly regulates the Ca²⁺ and Mn²⁺ concentrations at the Golgi level.

Results

Gdt1p mediates Mn²⁺ influx in L. lactis

To determine whether Gdt1p is able to transport Mn²⁺, we took advantage of the fact that the fluorescence emitted by Fura-2 is quenched by manganese (Fig. S1). The transport assays were carried out in the L. lactis DML1 strain, which was previously reported to be suitable for heterologous production of Gdt1p (4). More precisely, the fluorescence intensity was monitored over time on Fura-2–loaded L. lactis DML1 cells expressing GDT1 or containing the corresponding empty plasmid. The excitation wavelength was set at 360 nm, the Fura-2 isosbestic point at which its properties are independent on the Ca²⁺ concentration (Fig. S1), to reflect only Mn²⁺ transport. Addition of MnCl₂ at a concentration of 10 μm (Fig. 1A) and 25 μm (Fig. 1B) in the extracellular medium led to a significantly more pronounced decrease of the fluorescence signal over time for the cells producing Gdt1p compared with the ones transformed with the empty plasmid. Although the slight signal decrease observed for the negative control most likely reflects transport of Mn²⁺ by L. lactis endogenous transporters, the quenching observed to a greater extent for the clone producing Gdt1p indicates that it mediates Mn²⁺ influx in L. lactis. Interestingly, the Gdt1p-dependent quenching of the fluorescence emitted by Fura-2 occurred to a greater extent in the presence of 25 μm than in the presence of 10 μm manganese, thereby indicating a dose-dependent response. Similar transport measurements were carried out in the presence of 25 μm Co²⁺, Cu²⁺, and Ni²⁺, all three ions being also known to quench the fluorescence emitted by Fura-2. In those cases, no significant difference in signal decrease could be observed between the negative control and the cells producing Gdt1p (Fig. 1C), suggesting that these three ions are not transported by Gdt1p and strengthening that the effect observed for manganese on Fig. 1 (A and B) is well specific to Gdt1p.

Figure 1.

Gdt1p promotes Mn²⁺ influx in L. lactis. A, time-course measurement of the quenching by Mn²⁺ of the fluorescence emitted by Fura-2 in L. lactis DML1 cells transformed with the empty (C−) or GDT1-containing plasmid. The cells were grown to an A₆₀₀ of 0.5, at which expression of GDT1 was initiated by addition of 2.5 μg/liter nisin. After a 2-h postinduction time, the cells were washed and incubated for 2 h in the presence of Fura-2/AM. The fluorescence measurements were carried out in a calcium-depleted Tris buffer with an excitation and emission wavelength of 360 and 510 nm, respectively. Ten μm MnCl₂ were added in the extracellular medium after 60 s of measurement (indicated by the arrow). The data are represented as the mean fluorescence normalized to the fluorescence at time 0 (n = 3, ± S.D.). B, time-course measurement of the quenching by Mn²⁺ of the fluorescence emitted by Fura-2 in L. lactis DML1 cells in the presence of 25 μm MnCl₂ in the extracellular medium. The cells were treated, and the data are represented as described for A. C, normalized fluorescence recorded 5 min after addition of 25 μm MnCl₂, CoCl₂, CuCl₂, or NiCl₂ for the negative control (C−) and the clone producing Gdt1p. The cells were treated as described for A. The results are shown as the mean relative fluorescence ± S.D. (n = 3). ***, p < 0.001 (two-way analysis of variance with Bonferroni post hoc test).

Ca²⁺ and Mn²⁺ compete for transport by Gdt1p

We previously reported that Gdt1p mediates Ca²⁺ influx when produced in L. lactis (4). To highlight a putative competition between calcium and manganese for Gdt1p, we carried out transport assays in the presence of a fixed MnCl₂ concentration (25 μm) and variable CaCl₂ concentrations (0–50 μm) with an excitation wavelength at the isosbestic point of the probe (360 nm) to mainly reflect Mn²⁺ transport. As shown in Fig. 2A, the higher the CaCl₂ concentration, the lesser the quenching rate, most likely reflecting Mn²⁺ influx to a lesser extent. Inversely, the transport of Ca²⁺ (25 μm) was recorded in the presence of Mn²⁺ (0–100 μm) in a ratiometric mode (excitation wavelengths of 340 and 380 nm, emission wavelength of 510 nm), this latter being routinely used to monitor Ca²⁺ transport with Fura-2. In that case, the ratio of the fluorescence emitted at 510 nm after excitations at 340 and 380 nm (340/380 ratio), mainly reflecting the intracellular free Ca²⁺ concentration, increased to a lesser extent when the MnCl₂ concentration added in the extracellular medium increases (Fig. 2B). These results reinforce the conclusion that Ca²⁺ and Mn²⁺ are both transported by Gdt1p and suggest that these two cations compete for transport by Gdt1p. Additionally, it indicates that the protein is capable of transporting the two ions in the same direction under the conditions of gradients tested in this study.

Figure 2.

Ca²⁺ and Mn²⁺ influx are both promoted by Gdt1p in L. lactis. A, time-course measurements of the quenching by Mn²⁺ of the fluorescence emitted by Fura-2 in L. lactis DML1 cells producing Gdt1p in the presence of both MnCl₂ (fixed concentration of 25 μm) and CaCl₂ (variable concentration from 0 to 50 μm). Measurements were carried out with an excitation wavelength at the isosbestic point of Fura-2 (360 nm). The data are represented as the mean fluorescence normalized to the fluorescence at time 0. B, time-course measurements of the ratio of the fluorescence emitted at 510 nm after excitations at 340 and 380 nm (340/380) in the L. lactis DML1 clone in the presence of both CaCl₂ (fixed concentration of 25 μm) and MnCl₂ (variable concentration from 0 to 100 μm). In the two experiments, the cells were treated as described for Fig. 1A. MnCl₂ and CaCl₂ were added simultaneously after 60 s of measurement (arrow), and the curves are represented as means (n = 3) ± S.D.

Gdt1p diplays a higher affinity for Ca²⁺ than for Mn²⁺

To determine the affinity of Gdt1p for Ca²⁺ and Mn²⁺, we carried out in vivo transport measurements in L. lactis in the presence of various concentrations of these ions (from 2.5 to 200 μm CaCl₂ and from 5 to 500 μm MnCl₂). We then determined the initial slope of the fluorescence signal over time after addition of the cation for each concentration and plotted them according to the free substrate concentration. A Michaelis constant K_m was determined for each cation as the concentration at which half of the maximal initial slope was observed. Fig. 3A illustrates the time-course measurements at various CaCl₂ and MnCl₂ concentrations, from which the initial slopes were determined, whereas Fig. 3B shows the Michaelis–Menten curves that were fitted to the experimental data. The K_m calculated for Ca²⁺ and Mn²⁺ are respectively of 15.6 ± 2.6 and 83.2 ± 9.8 μm, suggesting that the affinity of Gdt1p toward Ca²⁺ is greater than the one toward Mn²⁺ in L. lactis.

Figure 3.

The affinity of Gdt1p for Ca²⁺ is higher than the one for Mn²⁺ in L. lactis. A, time-course measurements of the fluorescence emitted at 510 nm after excitations at 340 and 380 nm (340/380) (left panel) or of the fluorescence emitted at 510 nm after excitation at 360 nm normalized to the initial fluorescence (right panel) in L. lactis DML1 cells transformed with the GDT1-containing plasmid. The cells were treated as described for Fig. 1A. The indicated CaCl₂ (left panel) or MnCl₂ (right panel) concentration (μm) was added in the extracellular medium after 3 s of measurement. The data correspond to a representative set of three repetitions. B, values of the mean initial slopes (n = 3, ± S.D.) as a function of the free Ca²⁺ or Mn²⁺ concentration (left and right panels, respectively). The data were fitted to the Michaelis–Menten equation using GraphPad Prism.

Gdt1p is involved in yeast cellular manganese homeostasis

Our data suggest that Gdt1p could be a new actor involved in Mn²⁺ transport in yeast. To further assess the implication of Gdt1p in regulating yeast Mn²⁺ homeostasis, the total Mn²⁺ levels of the WT strain and of strains deleted for GDT1, PMR1, or both were evaluated using inductively coupled plasma atomic emission spectrometry (ICP–AES). The results, shown in Fig. 4A, indicate that deletion of GDT1 led to a 3.5-fold increase of the yeast total Mn²⁺ content compared with the WT. The pmr1Δ strain showed a higher cellular Mn²⁺ content, with a 20-fold increase compared with the WT. Interestingly, the gdt1Δpmr1Δ strain displayed a higher total Mn²⁺ content than the pmr1Δ strain. Additionally, expression of GDT1 from a plasmid in the gdt1Δ strain under the control of the endogenous promoter or of the strong TPI1 promoter partially or totally rescued the WT phenotype, respectively (Fig. 4B). The partial complementation by GDT1 under the control of its endogenous promoter correlates with an intermediate level of Gdt1p production, between the WT and the gdt1Δ strain, whereas the full complementation using the TPI1 promoter results from a higher level of Gdt1p production than in the WT. Using the same methodology, we previously reported Gdt1p-dependent effects on calcium accumulation in yeast (4). As a control, the intracellular levels of an ion not transported by Gdt1p, i.e. cobalt, were measured. No effect of the deletion of GDT1 could be observed for this ion (data not shown). These results support the conclusion that Gdt1p, in addition to Pmr1p, controls the total Ca²⁺ and Mn²⁺ content in yeast. To further evaluate the involvement of Gdt1p in Mn²⁺ tolerance, we monitored growth of the four strains in the presence or not of MnCl₂ in the extracellular medium. We could not detect any growth difference between the WT and the gdt1Δ strain when exposed to MnCl₂ concentrations of up to 10 mm, higher concentrations being lethal for the WT (data not shown). Interestingly, when exposed to 400 μm MnCl₂, a sublethal concentration for pmr1Δ, we identified a more pronounced growth defect for gdt1Δpmr1Δ than for pmr1Δ (Fig. 4C), thereby indicating that Gdt1p is involved in Mn²⁺ tolerance in a pmr1Δ background. These growth defects correlate with the fact that the highest Mn²⁺ accumulations were recorded in the pmr1Δ and double deletant strain. Most likely, Mn²⁺ accumulation in these strains reaches a threshold from which it becomes toxic for the cell. Similarly to what was observed for the ICP–AES analyses, expression of GDT1 under the TPI1 promoter could complement the growth defect of the gdt1Δpmr1Δ in the presence of manganese. Taken together, these results show that, in complement to Pmr1p, Gdt1p plays a role in yeast cellular manganese homeostasis.

Figure 4.

Gdt1p regulates the homeostasis of Mn²⁺ in yeast. A, cellular Mn²⁺ content of the WT, gdt1Δ, pmr1Δ, and gdt1Δpmr1Δ strains. The cells were grown in YD medium to an A₆₀₀ of 3, and the cellular Mn²⁺ content was measured by ICP–AES. The data are shown as means ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001 (unpaired t tests). B, left panel, cellular Mn²⁺ content of the WT and gdt1Δ strains transformed with the empty plasmid (Ø) or the plasmid containing GDT1 under the control of the TPI1 or of the endogenous GDT1 promoter. The strains were grown in MD−U medium to an A₆₀₀ of 3, and the cellular Mn²⁺ content was measured by ICP–AES. The data are shown as means ± S.D. (n = 3, excepted for gdt1Δ + pTPI-GDT1 where n = 2). ***, p < 0.001 (one-way analysis of variance with Bonferroni post hoc test). Right panel, production of Gdt1p in the corresponding strains. Total protein extracts of cells grown to an A₆₀₀ of 3 were analyzed by Western blotting with antibodies directed against Gdt1p. Coomassie Blue-stained polyvinylidene fluoride membranes were used as loading controls. C, growth of the WT, gdt1Δ, pmr1Δ, and gdt1Δpmr1Δ strains in rich solid medium supplemented or not with 400 μm Mn²⁺. The strains transformed with the empty (Ø) or GDT1-containing pRS416 plasmid were precultured in MD−U medium to an A₆₀₀ of 0.3. Serial 10-fold dilutions of the culture were dropped on YD solid medium containing or not 400 μm MnCl₂. The ΔΔ+GDT1 strain corresponds to the gdt1Δpmr1Δ mutant expressing GDT1 under the control of the constitutive TPI1 promoter. The plates were incubated 2 days at 28 °C.

Deletion of GDT1 restores the Sod2p activity in a smf2Δ background

Sod2p encodes a superoxide dismutase that localizes in the mitochondrial matrix and that catalyzes the breakdown of superoxide radicals to dioxygen and hydrogen peroxide. This enzyme requires Mn²⁺ as a cofactor (21). Luk and Culotta (22) previously reported that Smf2p, a yeast Nramp Mn²⁺ transporter found in intracellular Golgi-like vesicles (23), is required for acquisition by Sod2p of its cofactor. They additionally reported that the Sod2p activity defect was corrected when either growing cells in the presence of manganese or generating an increased intracellular manganese concentration through deletion of PMR1. In this study, we evaluated whether deletion of GDT1 could also restore the activity of Sod2p in the smf2Δ strain. To do so, cell lysates of the WT, smf2Δ, gdt1Δ, and pmr1Δ strains, as well as of the double deletants gdt1Δsmf2Δ and pmr1Δsmf2Δ, were loaded on a native polyacrylamide gel on which the Sod2p activity was monitored by staining with nitro blue tetrazolium (24, 25). As reported by Luk and Culotta (22), we observed a decreased activity of Sod2p in the smf2Δ strain that could be restored by deleting PMR1 and by growing cells with 40 or 60 μm MnCl₂ (Fig. 5, A and B). The activity of Sod2p could also be restored by the addition of MnCl₂ (40 mm) to the cell lysates, thereby indicating that the decreased activity of Sod2p does not result from in vivo protein degradation (Fig. 5C). Luk and Culotta (22) also verified by Western blotting that deleting SMF2 does not affect the abundance of Sod2p. Interestingly, whereas neither Gdt1p nor Pmr1p seem to be critical for delivery of manganese to Sod2p, deletion of GDT1 in the smf2Δ strain led to a Sod2p activity level similar to the one of the WT. This result shows that, like PMR1, deletion of GDT1 leads to a better bioavailability of Mn²⁺ for Sod2p, most likely through an increased cellular manganese content in the smf2Δ strain. This latter statement was verified by quantifying by ICP–AES the total manganese content of the strains for which the Sod2p activity was monitored. By doing so, we observed a 5- and 35-fold increase of the cellular manganese content, respectively, in the gdt1Δsmf2Δ and the pmr1Δsmf2Δ strain compared with the smf2Δ strain (Fig. 5D). Because the effects of deleting GDT1 or PMR1 are observed in the same direction both in terms of cellular manganese content and restoration of the Sod2p activity in an smf2Δ strain, this suggests that the direction of transport of Mn²⁺ is identical for these two proteins, i.e. from the cytosol to the Golgi.

Figure 5.

Effect of a gdt1Δ mutation on Sod2p activity and manganese accumulation in an smf2Δ mutant. A, activity of the Mn²⁺-dependent Sod2p in the indicated yeast strains. The seven yeast strains were grown to an A₆₀₀ of 3. Cell lysates were loaded on a native PAGE gel in which the activity of Sod2p was monitored by nitro blue tetrazolium staining. Colloidal blue-stained gels were used as loading controls. B, activity of Sod2p in the WT and smf2Δ strain grown in the presence of 0, 40, or 60 μm MnCl₂. C, activity of Sod2p in cell lysates containing 0, 20, or 40 mm MnCl₂. D, cellular Mn²⁺ content of the indicated strains. The cells were grown in YD medium to an A₆₀₀ of 3, and the cellular Mn²⁺ content was measured by ICP–AES. The data are shown as means ± S.D. (n = 3). **p < 0.01, ***p < 0.001 (unpaired t tests).

Discussion

Several recent studies suggested an implication of UPF0016 members in manganese homeostasis. However, no direct evidence of manganese transport by a UPF0016 member has been reported up to now. In this study, using transport assays in the bacterium L. lactis, we demonstrated that the yeast Gdt1p mediates Mn²⁺ transport across membranes. The role of Gdt1p in regulating manganese homeostasis was further shown in yeast. As supported by the competition assays carried out in this study, this Mn²⁺ transport activity adds up to the Gdt1p Ca²⁺ transport activity, which was previously demonstrated with a similar in vivo assay in L. lactis (4). Because Gdt1p was also reported to be involved in pH regulation (4, 26), we hypothesize that this protein acts as a Ca²⁺-Mn²⁺/H⁺ antiporter. However, stronger evidence for proton transport by Gdt1p still needs to be obtained.

This study therefore places Gdt1p as a novel protein implicated in both calcium and manganese yeast Golgi and/or cytosol homeostasis. The well characterized Golgi P-type ATPase Pmr1p is known to also regulate the homeostasis of these two ions at the Golgi level. Gdt1p and Pmr1p therefore most likely act in concert to correctly balance the concentration of Ca²⁺ and Mn²⁺ within the Golgi and the cytosol. However, our results suggest that the contribution of Gdt1p in regulating the yeast Mn²⁺ homeostasis is lower than the one of Pmr1p in the conditions tested in this study. Indeed, compared with a pmr1Δ strain, the absence of solely GDT1 does not cause any growth reduction in the presence of 400 μm Mn²⁺ and leads to a less pronounced increase of the total Mn²⁺ content. Additionally, a comparison of the affinity of Gdt1p for calcium and manganese cations estimated via the in vivo transport assays (K_m of respectively 15.6 and 83.2 μm) to the ones reported in the literature for Pmr1p (K_m of 0.1 μm for Ca²⁺ (27) and of 0.02 μm for Mn²⁺ (28)) highly suggests a higher affinity of Pmr1p for these two ions. To our knowledge, no data regarding the affinity of other yeast manganese transporters are currently available. However, we noticed that the affinity of Gdt1p for calcium is similar to the one reported for the vacuolar Ca²⁺/H⁺ antiporter Vcx1p (K_m of 25 μm (29)).

Although it is well known that Pmr1p transports Ca²⁺ and Mn²⁺ from the cytosol to the Golgi, the question of the direction of transport of these two ions by Gdt1p remains open. Provided that Gdt1p functions as a Ca²⁺-Mn²⁺/H⁺ antiporter, transport of Ca²⁺ and Mn²⁺ from the cytosol to the Golgi or in the opposite direction could both be considered. Gdt1p could also work reversely, depending on the neighboring gradients, or Ca²⁺ and Mn²⁺ could be transported in opposite directions. In this study, the effects of the absence of Gdt1p or Pmr1p on the cellular manganese content and on Sod2p activity in the smf2Δ strain were both observed in the same direction, thereby suggesting manganese transport in the same direction for these two proteins, from the cytosol to the Golgi. The increased cellular manganese content observed in the gdt1Δ and pmr1Δ strains would hence result from the inability to send Mn²⁺ to the Golgi lumen for further exit from the cell through secretory pathway vesicles. On the other hand, suppression of the Sod2p activity defects by deleting GDT1 or PMR1 in an smf2Δ background would result from an increased manganese concentration in the cytosol, from which it can be sent to the mitochondrial matrix. More generally, according to this hypothesis of direction of transport, Gdt1p would provide the Golgi with calcium and manganese cations and/or detoxify the cytosol in case of excess of these cations, in complement to Pmr1p. Based on the evaluation of the glycosylation process efficiency as an indicator of the Golgi Mn²⁺ concentration, the same direction of manganese transport was suggested by Dulary et al. (13). The entry of Mn²⁺ in the Golgi would then be controlled by an ATPase, Pmr1p, and by a secondary transporter, Gdt1p. Although the ATPases are generally associated with a high affinity but a low capacity of transport, the secondary transporters usually show a low affinity and a high capacity of transport. By comparing the K_m values obtained for Gdt1p to the ones reported in the literature for Pmr1p, we confirmed this trend in terms of affinity. The fact of having two transporters with different transport characteristics most likely enables a tight regulation of Mn²⁺ homeostasis at the Golgi level in a wide range of stresses and concentration gradients, thanks to a complementary action of the two transporters.

In either transport direction, Gdt1p is involved in the crucial regulation of both calcium and manganese, notably in the Golgi where part of the glycosylation process takes place. Because of the implication of the UPF0016 members, including the human TMEM165, in glycosylation, it is of specific interest to examine the link between Gdt1p and glycosylation in the light of its novel manganese transport activity. Indeed, Colinet et al. (4) previously reported Gdt1p-dependent glycosylation defects in yeast. More recently, Dulary et al. (13) analyzed the structural glycosylation abnormalities observed in a gdt1Δ strain cultured in the presence of a high external calcium concentration and reported defects in the α-1,3- and α-1,2-mannose branchings that correspond to deficiencies in late Golgi glycosyltransferases like Mnn2, Mnn5, and Mnn1. Interestingly, these three mannosyltransferases all require Mn²⁺ as a cofactor (18, 20). The Gdt1p-dependent glycosylation defects could be explained by the fact that, in the presence of high external Ca²⁺ concentration, Pmr1p would mainly transport Ca²⁺, thereby resulting in an excess of Ca²⁺ that could compete with Mn²⁺ for the cofactor binding site of the mannosyltransferases. This illustrates the importance of ensuring tight regulation of this Ca²⁺/Mn²⁺ ratio for proper protein maturation.

In conclusion, we propose that Gdt1p regulates the calcium and manganese yeast cytosolic and Golgi homeostasis in concert with Pmr1p, through its ability to transport both Ca²⁺ and Mn²⁺ cations. Our results suggest a direction of manganese transport from the cytosol to the Golgi. Confirming the direction of transport of Ca²⁺ and Mn²⁺ by Gdt1p would definitely constitute the next step toward a better understanding of its physiological role. Additionally, the results show for the first time direct Mn²⁺ transport by a UPF0016 member, thereby opening new ways to fully unravel the exact function of this protein family involved in essential cellular processes like photosynthesis in plants and cyanobacteria, as well as glycosylation in yeast and human.

Experimental procedures

Strains and culture media

The S. cerevisiae strains used in this study are listed in Table 1. Nontransformed yeast cells were routinely cultured at 28 °C in YD medium (2% yeast extract KAT, 2% glucose) under agitation. The cells transformed with plasmids were grown in MD minimal medium (0.7% yeast nitrogen base without amino acids (Difco), 2% glucose, supplemented with all amino acids except those used as a selection marker for plasmid maintenance). Solid media were produced by addition of 2% agar to the mixture. The L. lactis DML1 strain was kindly provided by B. Poolman (Groningen, Holland). L. lactis cells were grown in M17 broth according to Terzaghi (Merck Millipore) supplemented with 1% glucose at 28 °C. The cells transformed with the pNZ8048 plasmid were grown in the presence of 10 μg/ml chloramphenicol. Expression of genes under the control of the pNisA promoter was induced by 2.5 μg/liter nisin at the log phase (A₆₀₀ = ∼0.4–0.5).

Table 1.

Yeast strains used in this study

Strain	Description	Source
BY4741	Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0	Euroscarf
BY4741 gdt1Δ	Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 gdt1::KanMX4	Euroscarf
BY4741 pmr1Δ	Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 pmr1::KanMX4	Euroscarf
BY4741 smf2Δ	Mata his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 smf2::KanMX4	Euroscarf
BY4742 sod2Δ	MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 sod2::kanMX4	Euroscarf
BY gdt1Δ pmr1Δ	Matα his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 gdt1::KanMX4 pmr1::KanMX4	Ref. 3
BY smf2Δ pmr1Δ	Matα his3Δ1 leu2Δ0 ura3Δ0 smf2::KanMX4 pmr1::KanMX4	This study
BY smf2Δ gdt1Δ	Matα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 smf2::KanMX4 gdt1::KanMX4	This study

In vivo transport measurements

The in vivo transport measurements were carried out using the fluorescent dye Fura-2/AM according to the method previously described by Colinet et al. (4) with slight modifications. Briefly, L. lactis DML1 cells transformed with the empty or GDT1-containing pNZ8048 plasmid were grown in M17 broth. At an A₆₀₀ of 0.5, induction was initiated by adding 2.5 μg/liter nisin in the extracellular medium. After a postinduction time of 2 h, the cells were harvested (3,000 × g for 7 min) and washed twice with the washing buffer (50 mm Tris-HCl, pH 7.4, 100 mm KCl, 1 mm MgCl₂). The washed cells were resuspended in the same buffer supplemented with 0.2 mm EDTA (pH 8) and incubated for 10 min. The harvested cells were then incubated with the washing buffer supplemented with 10 μm Fura-2/AM and 1.7 mm probenecid for 2 h at 28 °C under agitation. The cells were subsequently washed twice in the presence of 1 mm EGTA, and the final pellet was resuspended in 10 ml of the washing buffer supplemented with 1.7 mm probenecid. Prior to measurements, this latter solution was treated with calcium sponges (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) chelator coupled to a polymer matrix; Invitrogen). The signal was recorded either with a single excitation wavelength of 360 nm (quenching measurements) or with two excitation wavelengths of 340 and 380 nm (ratiometric measurements), the emission wavelength being set at 510 nm. MnCl₂ or CaCl₂ were added in the extracellular medium at the indicated concentrations. The data were recorded using a JASCO FP8500 fluorometer controlled by the Spectra Manager software.

ICP–AES analyses

For determination of the total Mn²⁺ content of yeast mutant strains, the cells were grown in YD or minimal medium to an A₆₀₀ of 3. The cells were then collected by vacuum filtration using membrane filters (Millipore; 0.45-μm pore size) and washed twice with 2 ml of 1 mm EGTA (pH 8) and twice with 2 ml of H₂O. The cells were collected in heat-resistant beakers in 10 ml of H₂O and dried at 95 °C overnight and then in a dessicator for 24 h. The dry matter was mineralized by heating at 500 °C overnight. The ashed sample was subsequently dissolved in 10 ml 6.5% HNO₃ for analysis on an ICAP 6500 spectrometer (Thermo Scientific).

Western blotting

For Western blotting, 40 μg of proteins were separated on SDS-PAGE gels, and Western blotting was carried out as previously described (3). The primary rabbit antibodies against Gdt1p were previously produced in our lab (3). Horseradish peroxidase-coupled anti-rabbit IgG antibodies were purchased from IMEX.

Yeast drop tests

For drop tests, yeast cells were precultured overnight in 5 ml of minimal dextrose medium without uracil (MD−U). Each culture was then adjusted to an A₆₀₀ of 0.3. Four μl of the adjusted culture and of successive 10-fold dilutions were spotted on YD solid medium with or without 400 μm MnCl₂. The plates were incubated for 2 days at 28 °C.

Monitoring of Sod2p activity

To monitor Sod2p activity, the cells were grown in YD medium to an A₆₀₀ of 3 and washed before homogenization by glass-bead agitation in lysis buffer (10 mm sodium phosphate buffer, pH 7.8, 5 mm EDTA, 5 mm EGTA, 50 mm NaCl, 0.1% Triton X-100, 100 μg/ml PMSF, 4 μg/ml leupeptin, aprotinin, antipain, pepstatin, and chymostatin) (22). Proteins (200 μg) were loaded on a native PAGE gel, and migration was carried out at 100 V for 4 h. After migration, the gel was immersed in a 1 mg/ml nitro blue tetrazolium solution for 15 min, followed by an incubation in a solution containing 100 mm potassium phosphate buffer, pH 7.8, 28 mm tetramethylethylenediamine (TEMED), 2.8*10⁻² mm riboflavin, and 5 mm KCN for 15 min. The gel was finally exposed to light for about 30 min (24, 25). Colloidal blue gels were carried out in parallel as loading controls.

Author contributions

L. T., A. D., P. S., and P. M. conceptualization; L. T., A. D., and O. S. data curation; L. T. and A. D. formal analysis; L. T. validation; L. T. investigation; L. T. and A. D. methodology; L. T. writing-original draft; J. S. visualization; P. M. supervision; P. M. funding acquisition; P.M. writing-review and editing; L. T., A. D., and O. S. data acquisition.