Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 2019 Jun 12;180(4):2120–2132. doi: 10.1104/pp.18.01419

Dek40 Encodes a PBAC4 Protein Required for 20S Proteasome Biogenesis and Seed Development1

Guifeng Wang a,b,2, Wei Fan a,2, Mingyan Ou a, Xuewei Wang a, Hongli Qin a, Fan Feng a, Yulong Du a, Jiacheng Ni a, Jihua Tang b, Rentao Song c,3, Gang Wang a,d,3,4
PMCID: PMC6670095  PMID: 31189659

The PBAC4 chaperone encoded by Dek40 in maize influences 20S core protease biogenesis and is required for 26S proteasome function and seed development.

Abstract

The 26S proteasome, an essential protease complex of the ubiquitin-26S proteasome system (UPS), controls many cellular events by degrading short-lived regulatory proteins marked with polyubiquitin chains. The 20S proteolytic core protease (CP), the catalytic core of the 26S proteasome, is a central enzyme in the UPS. Its biogenesis proceeds in a multistep and orderly fashion assisted by a series of proteasome assembly chaperones. In this study, we identified a novel maize (Zea mays) kernel mutant named defective kernel40 (dek40), which produces small, collapsed kernels and exhibits delayed embryo and endosperm development. Dek40 was identified by map-based cloning and confirmed by transgenic functional complementation. Dek40 encodes a putative cytosol-localized proteasome biogenesis-associated chaperone4 (PBAC4) protein. DEK40 participates in the biogenesis of the 20S CP by interacting with PBAC3. Loss-of-function of DEK40 substantially affected 20S CP biogenesis, resulting in decreased activity of the 26S proteasome. Ubiquitylome analysis indicated that DEK40 influences the degradation of ubiquitinated proteins and plays an essential role in the maintenance of cellular protein homoeostasis. These results demonstrate that Dek40 encodes a PBAC4 chaperone that affects 20S CP biogenesis and is required for 26S proteasome function and seed development in maize.

The ubiquitin-26S proteasome system (UPS) participates in most proteolytic processes in eukaryotes. Abnormal proteins and short-lived proteins are recognized by the ubiquitin system and are marked with ubiquitin chains as degradation signals. Polyubiquitinated proteins are recognized and then degraded by 26S proteasomes. The UPS plays very important roles in most cellular processes such as cell-cycle control, DNA repair, signal transduction, cell death, immune responses, and metabolism (Glickman and Ciechanover, 2002; Wolf and Hilt, 2004; Demartino and Gillette, 2007; Tanaka, 2009).

The 26S proteasome, the central enzyme of the UPS, is composed of a proteolytic 20S core particle (CP) and one or two terminal 19S regulatory particles (RP; Coux et al., 1996; Baumeister et al., 1998; Finley, 2009; Budenholzer et al., 2017). The 20S CP is a barrel-shaped complex assembled by four stacked heptameric rings of related α- and β-subunits in a symmetric α1–7β1–7β1–7α1–7 configuration (Unno et al., 2002; Groll et al., 2005; Budenholzer et al., 2017). The 19S RP can be further dissected into two subparticles, the lid and base (Glickman, 2000; Park et al., 2010; Bhattacharyya et al., 2014; Collins and Goldberg, 2017). The 19S RP binds to one or both ends of the latent 20S CP to form an enzymatically active 26S proteasome.

The 20S CP is the catalytic core of the 26S proteasome. Its biogenesis occurs in an ordered set of steps initiating with formation of a heteromeric seven-membered α-ring (Ramos and Dohmen, 2008; Budenholzer et al., 2017). Because of the heteromeric nature of the α-ring in eukaryotes, different subunits must be incorporated in an exact order to ensure exact α-ring formation. Several eukaryotic α-subunits have retained the ability to form homomeric rings. For example, the human α7 subunit, upon expression in Escherichia coli, forms double ring structures, whereas α1 and α6 are unable to form such structures (Gerards et al., 1997). However, when expressed together with α7, they were also incorporated into the double ring assemblies, although in variable positions (Gerards et al., 1998). These observations suggest that not all α-subunits contain the necessary information for correct positioning within the α-ring, but instead most likely require additional guidance (Gerards et al., 1998). Indeed, emerging evidence indicates that assembly of the proteasomal subunits is not a spontaneous process but requires the help of a series of proteasome-specific chaperones (Rosenzweig and Glickman, 2008; Yashiroda et al., 2008; Murata et al., 2009; Sahara et al., 2014; Budenholzer et al., 2017).

The 20S CP biogenesis is mediated by at least four proteasome assembly chaperones 1–4 (PAC1–4) and UMP1/POMP in mammals, which are known as Poc1–4/Pba1–4 and Ump1 in yeast (Saccharomyces cerevisiae; Hirano et al., 2005, 2006; Le Tallec et al., 2007; Hoyt et al., 2008; Kusmierczyk et al., 2008; Rosenzweig and Glickman, 2008; Yashiroda et al., 2008; Murata et al., 2009; Matias et al., 2010; Sahara et al., 2014; Budenholzer et al., 2017). The biogenesis of the 20S CP begins with the α-ring formation supported by two heterodimeric chaperone complexes, such as PAC1–PAC2 and PAC3–PAC4 in mammals and Poc1–Poc2/Pba1–Pba2 and Poc3–Poc4/Pba3–Pba4 in yeast. The two chaperone complexes cooperate in the assembly of the α-ring and half-proteasomes. In yeast, Poc3–Poc4/Pba3–Pba4 functions at the early stage of α-ring assembly, starting with the formation of a Poc3–Poc4/Pba3–Pba4–α5 tertiary complex, assisting in the recruitment and ordering of neighboring α-subunits (Kusmierczyk et al., 2008, 2011; Yashiroda et al., 2008). The Poc1–Poc2/Pba1–Pba2 complex binds the α5 and α7 subunits and promotes formation of complexes containing all seven α-subunits (Hirano et al., 2005, 2006). In addition to promoting the assembly of α-rings, Poc1–Poc2/Pba1–Pba2 also suppresses α-ring dimerization, thereby promoting attachment of the β-subunits to the proper surface of the α-ring (Hirano et al., 2005, 2006). After completion of α-ring assembly, the β-subunits are incorporated onto the α-ring at their specific positions in a defined order. The precise mechanism of α-ring formation is unknown, but these chaperones play distinct and important roles at different stages during 20S CP biogenesis (Hirano et al., 2006).

Poc4/Pba4 is an assembly chaperone that was first found in yeast cells. Previous studies suggest that Poc4/Pba4 plays an important role in α-ring formation that occurs during the initial biogenesis of the 20S CP (Hirano et al., 2006; Le Tallec et al., 2007; Kusmierczyk et al., 2008; Yashiroda et al., 2008). The function of PAC4 (or Poc4/Pba4) has been documented partially in mammals and yeast (Le Tallec et al., 2007; Hoyt et al., 2008; Ramos and Dohmen, 2008). However, the exact mechanism of how Poc4/Pba4 (PAC4) affects 20S CP biogenesis, 26S proteasomal activity, and, particularly, its counterpart in plants, remains elusive. Here, we report the map-based cloning of Dek40 and demonstrate that it encodes a 20S proteasome biogenesis-associated chaperone4 (PBAC4). Loss-of-function of DEK40 impaired the enzyme activity of the 26S proteasome by affecting 20S CP biogenesis. Embryo and endosperm arrest resulted from disruption of DEK40 in maize (Zea mays). In this study, we confirm that defective PBAC4 affected seed development in maize by impairing 20S CP biogenesis in dek40.

RESULTS

dek40 Produces Small and Collapsed Kernels with Delayed Development

Compared with those of the wild type, the mutant dek40 kernels were small, flat, and slightly collapsed at maturity (Fig. 1A). The dek40 kernels were distinguished from their wild-type siblings as early as 12 d after pollination (DAP), characterized by the washy and top-pale appearance (Fig. 1B). Their genotypes were further confirmed by molecular markers 200211-4 and 213985-4, linked tightly with Dek40. The dek40 kernels became small and flat at 24 DAP (Fig. 1B). The 100-seed weight of mature dek40 kernels was only 42% of that of the wild-type kernels (Fig. 1C). The contents of major storage compounds at equal dry weight such as total starch, amylose, and total storage protein of mature kernels showed that there were no obvious differences between wild type and dek40 (Supplemental Fig. S1), but all compounds were reduced in dek40 when measured per kernel because of the equal dry flour weight derived from more dek40 kernels. After germination, the seedlings of dek40 exhibited slower growth and development than those of the wild type from the same ear (Fig. 1D). At 12 d after germination (DAG), the average plant height of dek40 seedlings was 9.8 cm, 30.7% of that of the wild type (31.9 cm; Fig. 1E), and the average root length of dek40 seedlings was 4.9 cm, 19.4% of that of the wild type (25.3 cm; Fig. 1F).

Figure 1.

Figure 1.

Open in a new tab

Phenotypic features of maize dek40 mutant. A, F2 ear of self-pollinated heterozygous dek40. The red arrows indicated dek40 kernels. Scale bar = 1 cm. B, Developing kernels phenotype of wild type and dek40 at 12 and 24 DAP. Scale bars = 1 cm. C, The 100-kernel weight of wild type and dek40. For each sample, three independent biological replicates were performed. n = 3 replicates. Error bars = average values ± sd. ***P < 0.001, Student’s t test. D, Phenotype of wild-type and dek40 seedlings at 12 DAG. Bar = 1 cm. E, Average plant height of wild-type and dek40 seedlings at 12 DAG. Error bars = average values ± sd. n = 20 seedlings per genotype; ***P < 0.001, Student’s t test. F, Average length of the primary root of wild-type and dek40 seedlings at 12 DAG. Error bars = average values ± sd. n = 20 seedlings per genotype. ***P < 0.001, Student’s t test. G, Light microscopy of 15- and 21-DAP embryos of the wild type and dek40. Scale bars = 200 μm. H, Light microscopy of 12- and 18-DAP endosperms of the wild type and dek40. Scale bars = 100 μm. I, SDS-PAGE analysis of zein in developing endosperms of wild type and dek40. The size of each band is indicated by numbers besides it. D, DAP. M, protein markers from top to bottom correspond to 34, 26, and 17 kD. WT, wild type.

Paraffin sections of wild-type and dek40 kernels segregating from the same ear at 15 and 21 DAP showed that the embryo of dek40 was morphologically normal but smaller (Fig. 1G) than that of the wild type. Resin sections of 12- and 18-DAP kernels showed that the mutant endosperm exhibited fewer starch granules than those of the wild type (Fig. 1H). The accumulation of the primary storage protein “zein” was delayed compared with that of the wild type during the endosperm development (Fig. 1I), indicating a substantially delayed development. By counting the number of cells in the 18 DAP endosperm, we found that the dek40 mutant had fewer cells than the wild type (reduced by 56%, Supplemental Fig. S2A). However, we also found that the mutant endosperm cells were larger than those in the wild type (increase of 155%, Supplemental Fig. S2B). These data indicated that development of embryo and endosperm of dek40 was delayed compared with that in the wild type.

Cloning of Dek40

After characterizing an F2 population with a total of 786 individuals using the molecular markers listed in Supplemental Table S1, Dek40 was mapped between the markers 200211-4 and InDel243-1-9 on chromosome 8, a region encompassing a physical distance of 1.6 Mbp (Fig. 2A).

Figure 2.

Figure 2.

Open in a new tab

Map-based cloning of Dek40. A, The Dek40 locus was mapped to a 1.6-Mbp region on chromosome 8. B, Structure, mutation site, and Mu insertion identification primers of the Dek40 gene. 1a, 019538-1a; 1s, 019538-1s; tir67, mu tir67. C, The Mu insertion was identified by PCR amplification using 1a, 1s, and tir67. The PCR product of W22 is only 769 bp (1a/1s). The PCR products of heterozygous are 769 bp (1a/1s), 412 bp (1a/tir67), and 491 bp (1s/tir67). The PCR products of dek40 are 412 bp (1a/tir67) and 491 bp (1s/tir67). D, RT-qPCR analysis of the Dek40 transcript in 12-, 18-, and 24-DAP endosperms of wild-type and dek40. For each sample, three independent biological replicates were performed. Error bars = average values ± sd. n = 3 replicates. ***P < 0.001, Student’s t test. The transcript levels of Dek40 were normalized to α-tubulin. E, Immunoblot analysis of DEK40 protein accumulation in 18-DAP endosperms of dek40, wild-type, and W22. WT, wild type.

The dek40 mutant originated from an active Mutator (Mu) line, and we supposed that the mutant was caused by an Mu insertion. Only one unique Mu insertion was found in this 1.6-Mbp interval. Flanking sequences analysis and PCR amplification indicated that a Mu insertion occurred in the −540 bp upstream of the ATG start codon of GRMZM2G019538 (Fig. 2, B and C). Transcript expression analysis revealed that expression of GRMZM2G019538 was downregulated in dek40 (Fig. 2D; Supplemental Fig. S3). Thus, GRMZM2G019538 was considered as the candidate gene.

Using an antibody raised against the protein encoded by GRMZM2G019538, we detected a protein with an expected molecular mass of 17 kD in developing wild-type and W22 kernels, whereas almost no protein was detected in dek40 (Fig. 2E). This result indicated the Mu insertion in dek40 caused a dramatic decrease in GRMZM2G019538 protein accumulation.

Complementation Analysis

A functional complementation test was used to confirm the cloned candidate gene. Complementation assays were performed using the validated 4,138-bp genomic DNA (gDNA) fragment of Dek40 (GRMZM2G019538) containing the entire coding region, a 1,932-bp upstream sequence, and a 891-bp downstream sequence amplified from B73 genomic DNA. After transforming into the Hi-II hybrid (parent A [pA] and parent B [pB]), four independent T0 transgenic lines were obtained and crossed to dek40 heterozygous plants. Both transgene hemizygous and dek40 heterozygous kernels from two single copy T1 lines were planted and self-crossed for further analysis (Fig. 3A). The ratio of the segregating small and collapsed kernels was drastically reduced to ∼6% (1:16), far less than the 25% (1:4) ratio expected otherwise. After genotyping and transgene detection, the kernels were classified into several classes. For the dek40 homozygous seeds, only the transgenic ones displayed the wild-type phenotype. The kernel size, average plant height, and average root length of transgenic lines displayed the wild-type phenotype, but those that were nontransgenic were similar to the mutant (Fig. 3, B–E). Reverse transcription quantitative PCR (RT-qPCR) and western blot analysis revealed that GRMZM2G019538 expression of transgenic lines also recovered to the levels of the wild type but that it did not occur for those nontransgenic ones (Fig. 3, F and G). These results indicated that the dek40 phenotype was fully rescued by the transgenic candidate gene. Therefore, the dek40 allele was functionally complemented by the wild-type allele through stable genetic transformation. These results confirmed that the candidate gene GRMZM2G019538 identified by map-based cloning was the Dek40 gene.

Figure 3.

Figure 3.

Open in a new tab

Transgenic confirmation of Dek40. A, Self-crossed T2 ear of the transgene hemizygous and dek40 heterozygous T1 lines. B, Phenotype of 20 wild-type kernels (upper and middle) and 10 dek40 mutant kernels (lower) obtained from a segregated transgenic line. C, Molecular analysis of transgenic kernels of the complementation test. Genotyping the 30 kernels for Dek40 locus on chromosome 8 using the molecular marker 200211-4 (upper) and 213985-4 (middle). 1, H2O control; 2, pApB; 3, dek40; M, marker; transgene detection of the 30 kernels using the marker pTF102-ORF-F1R1 (lower). 1, H2O control; 2, pApB; 3, pTF102-DEK40 vector DNA; M, marker. D, Phenotype of different kinds of seedlings at 12 DAG. Scale bars = 1 cm (A, B, and D). E, Average plant height and primary root length of different kinds of seedlings at 12 DAG. F, RT-qPCR analysis of the Dek40 transcript in different kinds of kernels at 12, 18, and 24 DAP. The transcript levels of Dek40 were normalized to α-tubulin. G, Immunoblotting analysis of the DEK40 protein in different kinds of kernels at 12, 18, and 24 DAP. E, n = 20 seedlings per genotype. In (F), for each sample, three independent biological replicates were performed. n = 3 replicates, error bars = average values ± sd. *P < 0.05, ***P < 0.001, Student’s t test. The transcript levels of Dek40 were normalized to α-tubulin. From (C) to (G), WT−, nontransformed wild-type endosperms; dek40+, transformed and dek40/dek40 homozygous endosperms; dek40−, nontransformed and dek40/dek40 homozygous endosperms.

Dek40 Encodes a Cytosol-Localized PBAC4

The genomic DNA sequence of GRMZM2G019538 spans 2.5 kb and contains five exons and four introns (Fig. 2B). We obtained the full-length complementary DNA of the Dek40 gene of B73 by rapid-amplification of complementary DNA ends. We found that the transcript length was 1,175 bp and contained a 486-bp coding sequence and noncoding regions of 259 and 430 bp at the 5′ and 3′ ends, respectively (Fig. 2B). GRMZM2G019538 encoded a 17-kDa protein of 161 amino acids. BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) searches, using the DEK40 amino acid sequence as a query, indicated that it was a putative PAC4. We constructed a phylogenetic tree on the basis of the DEK40 protein sequence from Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), sorghum (Sorghum bicolor), maize, and PAC1–4 from humans. The phylogenetic tree and alignment suggested DEK40 is more like that of PAC4 in sorghum and rice than that in human (Fig. 4A). A detailed sequence alignment among these PAC4 homologs from Arabidopsis, rice, sorghum, maize, humans, and yeast showed that DEK40 harbored the conserved domain of PAC4 (Fig. 4B). We named it “PBAC4.”

Figure 4.

Figure 4.

Open in a new tab

Dek40 encodes a cytosol-localized PBAC4. A, Phylogenetic relationships of DEK40 and its homologs. B, Alignment of the deduced amino acid sequence of PAC4 with selected homologous proteins. The conserved residues of PAC4 are highlighted in red. C, DEK40 is mainly localized to the cytosol. Fractions were subjected to immunoblot analysis with antibodies against DEK40, BIP (heavy-chain binding protein, membrane marker), and Histone 3 (H3, nucleus marker). In (A) and (B), Zm, Z. mays; Sb, S. bicolor; Os, O. sativa; At, Arabidopsis; Hs, H. sapiens; Sc, S. cerevisiae.

Cell fractionation was used to detect the presence of DEK40 in subcellular fractions using a DEK40 polyclonal antibody. Proteins extracted from 18-DAP kernels of W22 were separated into soluble and organelle fractions. DEK40 was detected in the soluble fraction, indicating that DEK40 was primarily localized to the cytosol (Fig. 4C).

Dek40 Is Constitutively Expressed

RT-qPCR analysis revealed that Dek40 was expressed widely in root, stem, leaf, husk, silk, tassel, ear, sheath, and kernel (Fig. 5A). The strongest expression was detected in ears. Because the dek40 mutant phenotype is related to maize kernels, we further examined the expression profile of Dek40 during kernel development. The Dek40 transcript was first detectable at 3 DAP and increased in abundance, peaking at 5 DAP (Fig. 5A). A relatively low amount of Dek40 expression was detected from 7 DAP to 33 DAP (Fig. 5A). DEK40 protein accumulation in developing kernels was consistent with the RNA level, showing that Dek40 was expressed in different developmental stages of maize kernels (Fig. 5B).

Figure 5.

Figure 5.

Open in a new tab

Expression pattern of the Dek40 gene. A, RT-qPCR analysis of Dek40 transcript in root, leaf, stalk, tassel, embryo, and developing endosperms from 3 DAP to 33 DAP. The transcript levels of Dek40 were normalized to α-tubulin. For each sample, three independent biological replicates were performed. Error bars = average values ± sd. n = 3 replicates. B, Immunoblot analysis of DEK40 protein accumulation in wild-type seeds at different developmental stages. WT, wild type.

DEK40 Interacts with Maize PBAC3

In yeast and humans, Poc4/Pba4 or PAC4 plays an important role in 20S CP biogenesis by interacting with Poc3/Pba3 or PAC3 (Le Tallec et al., 2007; Kusmierczyk et al., 2008, 2011; Yashiroda et al., 2008). We verified the protein interaction between DEK40 (GRMZM2G019538) and maize PBAC3 (GRMZM2G028902) by the yeast two-hybrid assay. The open reading frame (ORF) of Dek40 was fused in frame with the GAL4 DNA binding protein. This bait, without self-activation, allowed us to test the interaction between DEK40 and the full-length PBAC3 protein. As shown in Figure 6A, we found that DEK40 interacted with PBAC3. To further explore the interaction relationship between DEK40 and PBAC3 in vivo, a bimolecular luciferase complementation (BiLC) experiment was also performed in which DEK40 and PBAC3 were fused to the N- and C-terminal domains of luciferase (NLUC and CLUC, respectively). Agrobacterium harboring the DEK40-NLUC and PBAC3-CLUC constructs was infiltrated into 3-week–old Nicotiana benthamiana leaves. The result showed that cotransfection of DEK40-NLUC and PBAC3-CLUC produced strong luciferase activity, whereas DEK40-NLUC and the corresponding empty construct CLUC failed to produce a visible signal (Fig. 6B; Supplemental Fig. S4).

Figure 6.

Figure 6.

Open in a new tab

DEK40 interacts with PBAC3. A, Yeast two-hybrid assay showing that DEK40 and PBAC3 interact. AD, activating domain; BD, binding domain. B, BiLC showing that DEK40 and PBAC3 interact. Fluorescence signal intensities represent their interaction activities.

DEK40 Is Required for 20S CP Biogenesis

To ascertain whether DEK40 is responsible for 20S CP biogenesis, 26S proteasomes and proteasome subcomplexes were isolated from 18 DAP kernels of wild type (Le Tallec et al., 2007; Book et al., 2010; Li et al., 2015a). A total of 20 samples were collected from the fraction collector. The different fractions from 9 to 18 were separated by SDS-PAGE and analyzed by western blotting analysis using antiα3, antiβ2, and anti-DEK40 antibodies. The result showed that DEK40 was mainly located in the components (fractions 12–16, ≤ 670 kD) that were <20S CP (670–720 kD; Fig. 7A).

Figure 7.

Figure 7.

Open in a new tab

DEK40 affects 20S CP biogenesis and 26S proteasome activity. A, Proteasome complexes extracted from 18-DAP endosperms of wild type were analyzed by immunoblotting using anti-DEK40, antiα3, and antiβ2 antibodies. B, The relative abundances of double-capped (RP2-CP), single-capped (RP1-CP), and 20S CP proteasomes in 18-DAP endosperms of wild type and dek40 mutant were examined by native PAGE in conjunction with immunoblotting using antiβ2 antibody. C and D, Activity analysis of 26S proteasome in 12- and 24-DAP kernels of wild type and dek40. RFU, relative fluorescence units. For each sample, three independent biological replicates were performed. Error bars = average values ± sd. n = 3 replicates. ***P < 0.001, Student’s t test. WT, wild type.

To substantiate whether disruption of DEK40 affected the 20S CP biogenesis, the mixture of size-fractionated extracts (1–12, ≥ 670 kD) from 18-DAP kernels of wild type and dek40 was separated using Blue Native-PAGE and then analyzed by immunoblotting using antiβ2 antibody and anti-Rpt5 antibody, respectively. The immunoblot results of antiβ2 antibody and anti-Rpt5 antibody showed that the amount of the 30S (RP2+CP) proteasome was comparable between wild type and dek40, but the 26S (RP1+CP) proteasome was decreased in dek40 (Fig. 7B; Supplemental Fig. S5). The immunoblot results of antiβ2 antibody revealed that dek40 lost nearly all free 20S CP (Fig. 7B). The anti-Rpt5 antibody analysis revealed that free 19S RP accumulated in dek40. These results demonstrated that DEK40 is required for efficient 20S CP biogenesis in maize.

dek40 Exhibits Impaired Enzyme Activity of 26S Proteasome

To investigate the enzyme activity of the 26S proteasome corresponding to the inefficient 20S CP biogenesis in dek40, we tested the Suc-LLVY–hydrolyzing activities of the mixtures of size-fractionated extracts (1–12, ≥ 670 kD) from 12- and 24-DAP kernels of the wild type and dek40 using the specific fluorogenic substrates (Smalle et al., 2002; Li et al., 2015b). Remarkably, a reduction of proteolytic activities in fractions containing both 20S and 26S proteasomes was observed in extracts from dek40 compared with those of the wild type (Fig. 7, C and D). The proteasomes activity in dek40 was reduced by 20% to 55% compared with that of the wild type (Fig. 7, C and D). Because proteasome impairment is expected to increase ubiquitin-substrate levels, we analyzed the ubiquitinated proteins by anti-ubiquitin antibody. Immunoblot analysis showed that the accumulation of polyubiquitinated proteins was elevated in dek40 (Supplemental Fig. S6).

Ubiquitylome Analysis of Wild-Type and dek40 Kernels

Generally, the degradation of ubiquitinated proteins is affected when proteasome functions were impaired. To confirm this relationship, the ubiquitylome of 15 DAP kernels of the wild type and dek40 was investigated by immunoprecipitation coupled with tandem mass spectrometry (IP-MS/MS) using the method described in Figure 8A. After analyzing three biological replicates for the wild type and dek40, 2,234 total Lys ubiquitination (Kub) sites in 1,024 proteins were identified in wild-type and dek40 (IPX0001367000, http://www.iprox.org), among which 1,367 sites in 708 proteins were accurately quantified, possessing consistent quantification ratios in at least two replicates. We observed that ubiquitination levels of 481 sites in 384 proteins were differential between wild type and dek40 using a 2.0-fold threshold (P < 0.05; Supplemental Dataset S1). Of these sites, 270 sites in 221 proteins were upregulated, and 211 sites in 167 proteins were downregulated in dek40 (P < 0.05; Supplemental Dataset S1).

Figure 8.

Figure 8.

Open in a new tab

Differentially ubiquitylated proteins analysis in wild type and dek40. A, Schematic diagram of ubiquitin-modified proteome characterization protocol (LC-MS/MS). B and C, GO-based enrichment analysis of proteins with upregulated (B) and downregulated (C) Kub sites in dek40. The percentage of differentially ubiquitylated proteins indicates the ratio of the enriched proteins to all upregulated or downregulated proteins. D, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-based enrichment analysis of proteins with upregulated and downregulated Kub sites in dek40.

To understand the cellular function of the differentially ubiquitinated proteins, Gene Ontology (GO) annotations were used to classified the proteins according to their involvement in biological processes, molecular functions, and cellular components (Fig. 8, B and C; Supplemental Dataset S1). In terms of biological processes, the upregulated proteins in dek40 were primarily divided into metabolic process, gene expression, translation, proteolysis, ubiquitin-dependent protein catabolic process, development process, cell division, and response to hormone stimulus (Fig. 8B), whereas the downregulated proteins in dek40 were divided into different metabolic processes and biosynthetic processes (Fig. 8C). Molecular functions analysis showed that the upregulated proteins in dek40 were enriched in hydrolase activity, catalytic activity, structural molecular activity, peptidase activity, DNA binding activity, and ubiquitin-like modifier-activating enzyme activity (Fig. 8B). The downregulated proteins in dek40 were enriched in catalytic activity, transferase activity, cofactor binding activity, lyase activity, translation elongation factor activity, and oxidoreductase activity (Fig. 8C). To determine the cellular components into which the differential ubiquitinated proteins distributed in maize cells, we classified these proteins into different categories. GO analysis showed that the upregulated proteins in dek40 were located in the following categories: cytoplasm, different complexes (such as macromolecular, protein, catalytic, endopeptidase, proteasome complex), nucleus, and ribosome (Fig. 8B). The downregulated proteins in dek40 were located in actin cytoskeleton (Fig. 8C).

KEGG pathway analysis was used to reveal the metabolic processes of these differentially ubiquitinated proteins in maize. The results showed that the proteins were primarily involved in the following 11 metabolic pathways (Fig. 8D; Supplemental Dataset S1): ribosome, proteasome, protein-processing pathways in the endoplasmic reticulum, glycolysis/gluconeogenesis, spliceosome, carbon fixation in photosynthetic organisms, pyruvate metabolism, RNA transport, ubiquitin-mediated proteolysis, amino sugar and nucleotide sugar metabolism, and Trp metabolism (Fig. 8D; Supplemental Dataset S1).

DISCUSSION

Dek40 Encodes a PBAC4 in Maize

In both mammals and yeast, PAC1–4 and UMP1 or Poc1–4/Pba1–4 and Ump1 are involved in the biogenesis of the 20S CP. These chaperones are required for proper subunit–subunit contacts in 20S CP biogenesis. However, none of the chaperones have been analyzed in plants. In this study, Dek40 was isolated by map-based cloning and was predicted to encode PAC4. Phylogenetic tree and alignment suggested DEK40 is more like that of PAC4 in sorghum and rice than that in human (Fig. 4, A and B). Previous studies in yeast and mammals confirmed that a heterodimeric complex of PAC3 and PAC4 is involved in 20S CP biogenesis. Both yeast two-hybrid and BiLC assays confirmed that DEK40 interacted with PBAC3 (Fig. 6, A and B; Supplemental Fig. S4). These results indicated that Dek40 encoded a PBAC4 that could interact with PBAC3 in maize.

Chaperone-Dependent Proteasome Biogenesis Is an Important (But Not the Only) Pathway in Maize

The 20S CP biogenesis is mediated by Poc1–4/Pba1-4 or PAC1–4 in yeast or mammals. The biosynthesis of 20S CP will be affected by the knockdown of any one of PACs in yeast and mammalian. In mammals, the amount of the 20S CP in PAC1-deficient livers was only 20% to 30% of the control liver (Sasaki et al., 2010). The authors speculated that >70% to 80% of the 20S proteasome was generated in a PAC1-dependent manner and that only a small amount of the 20S proteasome was generated without PAC1 (Sasaki et al., 2010). Multicellular eukaryotic organisms often express different 20S proteasome subtypes (Ramos and Dohmen, 2008). For example, two types of vertebrate-specific and best-studied proteasome subtypes, immunoproteasome and thymoproteasome, are found in vertebrates (Murata et al., 2001, 2007; Ramos and Dohmen, 2008). In Drosophila, a testis-specific proteasome subtype is essential for spermatogenesis (Zhong and Belote, 2007). In both rice and Arabidopsis, most, if not all, of the CP and RP subunit pairs are expressed and assembled into the mature 26S particle, implying that plants generate a wide assortment of proteasome types (Shibahara et al., 2004; Yang et al., 2004). These observations indicated that functionally specialized alternative 20S proteasome subtypes might also exist in plants (Ramos and Dohmen, 2008; Book et al., 2010). In this study, we found that dek40 lost almost all free latent 20S CP. However, dek40 contained normal amounts of the 30S proteasome (RP2+CP) and less of the 26S proteasome (RP+CP; Fig. 7B). The origin of the 20S CP involved in the assembly of 30S and 26S proteasomes remains unclear. These data suggested that alternative biogenesis pathways independent of PACs most likely also exist in maize. Importantly, the PAC-dependent pathway is the major route to generate the 26S proteasome in maize. These facts also showed that functionally specialized alternative 20S proteasome subtypes might exist in maize and other plants. However, the components required for the biogenesis of these alternative 20S proteasome subtypes remain elusive.

DEK40 Impaired 26S Proteasome Activity by Affected 20S CP Biogenesis

The assembly of the proteasomal subunits requires the help of a series of proteasome-specific chaperones to ensure that every subunit occupies a particular, predetermined place within the newly synthesized proteasome (Sahara et al., 2014). However, these chaperones have not been identified and characterized in plants. In this study, Dek40 encoded a cytosol-localized PBAC4 chaperone that was isolated by map-based cloning in maize. DEK40 participated in the biogenesis of the 20S CP by interacting with PBAC3 (Figs. 6A and 7A). In mammals, the knockdown of the assembly chaperones affects 20S CP biogenesis and therefore reduces 26S proteasome activity (Hirano et al., 2006, 2008; Kaneko et al., 2009). Loss-of-function of DEK40 affected 20S CP biogenesis, resulting in decreased activity of the 26S proteasome. Generally, most mutants arrested either in proteasome biogenesis or function are consequently defective in the degradation of high-molecular–weight ubiquitin conjugates. Our result indicated that the accumulation of polyubiquitinated proteins was elevated in the dek40 mutant (Supplemental Fig. S6), caused by the decreased proteasome activity in dek40, leading to the impaired degradation of ubiquitinated proteins.

Protein Ubiquitination Affects Various Aspects of Seed Development in Maize

Previous studies demonstrated that protein ubiquitination is a major posttranslational modification that regulates various aspects of plant biology, such as metabolism, cell division, cellular transport, signal transduction, transcription, translation, and endocytosis (Muratani and Tansey, 2003; Wagner et al., 2011; Kim et al., 2013; Xie et al., 2015; Guo et al., 2017). Plants also depend on protein ubiquitination and proteolysis conducted by the UPS to control the abundance of these regulatory proteins and enzymes. Thus, changes in 26S proteasome activity will affect the degradation of ubiquitinated proteins and cellular protein homoeostasis. The ubiquitylome of 15-DAP kernels indicated that ubiquitination levels of 481 sites in 384 proteins were differential between the wild type and dek40 (Supplemental Dataset S1). These proteins were involved in various cellular processes, including metabolism, gene expression, translation, protein catabolism, development, and cell division. These results suggest that protein ubiquitylation plays important roles in protein degradation and influences a wide range of physiological and developmental processes in maize.

GO and KEGG analysis showed that these differentially ubiquitinated proteins between wild type and dek40 were involved in several vital metabolic pathways (Fig. 8, B–D). First, we found that 15 differentially ubiquitinated proteins were enriched in glycolysis/gluconeogenesis, carbon fixation pathway in photosynthesis, and pyruvate metabolism pathways (Fig. 8, B–D; Supplemental Dataset S1). These proteins play important roles in starch and Suc metabolism. Resin sections of 12- and 18-DAP kernels indicated that dek40 endosperms had fewer starch granules than those in the wild type of the same stage (Fig. 1H). Therefore, differentially ubiquitinated proteins affected starch synthesis of dek40. Second, GO and KEGG analysis showed that 33 differentially ubiquitinated proteins were involved in gene expression, translation, and protein metabolism and catabolism (Fig. 8, B–D; Supplemental Dataset S1). SDS-PAGE analysis demonstrated that the accumulation of the primary storage protein zein was delayed in dek40 immature kernels. The data suggested that these proteins affected protein synthesis and accumulation in dek40. Finally, we identified 20 differentially ubiquitinated proteins associated with cell division (Fig. 8, B–D; Supplemental Dataset S1). In the past few years, many proteins necessary for cell division have been discovered to undergo processing and functional limitation by entering the UPS pathway with the final destination to be degraded by the 26S proteasome (Adams, 2004; Goldberg, 2007). Resin sections of immature kernels confirmed that dek40 had fewer cells and cells with larger volume than those of the wild type (Supplemental Fig. S2). This finding is reminiscent of the compensation phenomenon observed in many mutants with reduced cell division in which the plant attempts to reach a normal tissue size by increasing cell expansion (Horiguchi et al., 2006). Paraffin and resin sections of immature kernels also confirmed that development of both endosperm and embryo was slower than that of the wild type. Collectively, the defective biogenesis of 20S CP caused by dysfunction of DEK40 impaired the 26S proteasome activity, thereby affecting the degradation of ubiquitinated proteins. The disorder in ubiquitinated protein degradation would slow down the accumulation of cell contents, cell division, and development in endosperm and embryo, ultimately generating small, flat, and collapsed mutant seeds.

MATERIALS AND METHODS

Plant Materials

The maize (Zea mays) dek40 mutant was obtained from the Maize Genetics Cooperation Stock Center (stock no. 5511G), and was crossed into the W64A genetic background with five rounds of backcrosses. The mutant was crossed into the W64A to produce the F2 population. Seeds of the Hi-II pA and Hi-II pB lines for maize genetic transformation were initially obtained from the Maize Genetic Cooperation Stock Center and amplified on the campus of Shanghai University. The F2 immature zygotic embryos (1.5–2.0 mm) of the maize Hi-II hybrid genotype were aseptically dissected from ears harvested 10–12 DAP and were used for genetic transformation (Wang et al., 2011). Maize plants were cultivated in the field at the campus of Shanghai University.

Light Microscopy

For light microscopy, immature dek40 and wild-type kernels were obtained from the ear of a self-crossed heterozygous plant. Samples were cut along the longitudinal axis into three equal parts and the central slice was used as described previously by Wang et al. (2014).

Measurement of Starch and Proteins

At least 20 mature kernels or 30 immature kernels of the wild type and dek40 were ground into fine flour. One-hundred milligrams of flour for each sample was used for measurement of starch and proteins. The protocols for these measurements were performed according to Wang et al. (2011), and measurements were replicated three times for wild-type and dek40 endosperm. Equal volume of zein, nonzein, and total proteins extracted from equal weight flour were separated by SDS-PAGE to analyze the accumulation patterns in wild-type and dek40 kernels (Wang et al., 2011).

Map-Based Cloning and Mu Tag Isolation

The dek40 locus was mapped using an F2 population of 786 mutant plants in the W64A background. Molecular markers distributed throughout maize chromosome 8 were utilized for preliminary mapping. Molecular markers for fine mapping (Supplemental Table S1) were developed to narrow the locus to a 1.6-Mbp region. Mu tag isolation was performed according to Williams-Carrier et al. (2010).

Complementation Test

For the functional complementation test, a validated 4,138-bp genomic DNA fragment containing the entire coding region, a 1,932-bp upstream sequence, and a 891-bp downstream sequence were amplified from B73 gDNA using DEK40FL-gDNA F1 and DEK40FL-gDNA R4 primers (Supplemental Table S1). The gDNA was cloned into pTF102, which carried a bar resistance marker, using EcoRI and SacI. The resulting construct (pTF102-DEK40) was transformed into maize immature zygotic embryos of the Hi-II hybrid seeds of the Hi-II pA and Hi-II pB lines. The positive T0 lines were crossed to dek40 heterozygous plants for further analysis. For the molecular identification of transgenic plants, a marker was designed to identify the Dek40 gene with the primer pair pTF102-F1 and pTF102-R1 (Supplemental Table S1). The markers 200211-4 and InDel243-1-9 (Supplemental Table S1), linked tightly with the Dek40 locus, were designed to identify the genotype of the dek40 mutant.

RNA Extraction and RT-qPCR

Root, stem, the third leaf, silk, tassel, ear, husk, and sheath tissues were collected from at least three W22, at the V12 stage, and immature seeds were harvested at 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 DAP. Immature kernels from the wild-type, transgenic homozygote dek40/dek40 and nontransformed homozygote dek40/dek40 were harvested at 12, 18, and 24 DAP for further analysis. RNA extraction, purification, and quantification were performed according to Wang et al. (2012). The RT-qPCR experiments were performed to three independent RNA samples sets with α-tubulin as the reference gene. From a pool of kernels collected from three individual plants, each RNA sample was extracted, for which three technical replicates were performed according to Wang et al. (2014). The RT-qPCR primers are listed in Supplemental Table S1.

Phylogenetic Analysis

Related sequences were identified in the NCBI nonredundant protein sequences database by performing a BLASTP search (Camacho et al., 2009) with the maize DEK40 protein sequence. The phylogenetic tree of PAC4 of maize (DEK40), sorghum (Sorghum bicolor; SbPAC4), rice (Oryza sativa; OsPAC4), Arabidopsis (Arabidopsis thaliana; AtPAC4), and humans (HsPAC4) was constructed using the program MEGA v7.0 (www.megasoftware.net). The evolutionary history was inferred using the minimum evolution method. The bootstrap consensus tree inferred from 1,000 replicates is taken to represent the evolutionary history of the taxa analyzed (Kumar et al., 2016). The reference sequences were HsPAC1, HsPAC2, and HsPAC3 (PAC1–3) of humans. Amino acid sequences of PAC4 of maize, sorghum, rice, Arabidopsis, humans, and yeast (Saccharomyces cerevisiae; ScPAC4) were aligned with the MUSCLE method in the software program MEGA v7.0 (www.megasoftware.net) using their default. Settings were for protein multiple alignments.

Production of Polyclonal Antibodies

The full-length Dek40 ORF was amplified from cDNA of W22 endosperm using primers DEK40-ORF-EcoRI-F and DEK40-ORF-SalI-R (Supplemental Table S1), digested with EcoRI and SalI, and cloned into pGEX-4T-1 (Amersham Biosciences) to create an in-frame fusion with glutathione S-transferase (GST). Theα3 or β2 protein fused with GST was also used as antigen (primers are listed in Supplemental Table S1). Antibodies were produced in white rabbits by ABclone Technology (http://abclonal.com.cn/).

Subcellular Fractionation, Total Endosperm Protein Extraction, and Immunoblot Analysis

For subcellular fractionation, 18-DAP kernels of W22 were ground into a powder in liquid nitrogen and dissolved in resuspension buffer (Wang et al., 2014). After removing cell debris by centrifuging at 500g and 10,000g, total proteins were separated into soluble and total cell organelle fractions according to Wang et al. (2014). Nuclear protein fractionation was performed according to Qi et al. (2016).

Total protein from wild-type and dek40 endosperms was extracted according to the method of Wang et al. (2014). Protein separation, transfer, and immunoblotting were according to Wang et al. (2014). Immunoblot analysis with ubiquitin antibody (Cayman Chemical Company; 1:300 dilution), DEK40 antibody, α3 antibody, and β2 antibody (1:500 dilution) and BiP antibody (Santa Cruz Biotechnology), Histone antibody (Cell Signaling), Rpt5a/b antibody (PHYTOAB), and α-tubulin antibody (Sigma-Aldrich; 1:1000 dilution) was performed according to the manufacturer’s instructions.

Protein Interaction Experiments

The ORF of Dek40 amplified by DEK40-ORF-EcoRI-F and DEK40-ORF-BamHI-R (Supplemental Table S1) was fused downstream to the GAL4 BD domain in pGBKT7 at the EcoRI and BamHI sites. The ORF of PBAC3 was cloned from maize and inserted in frame into pGADT7. Yeast transformation and screening procedures were performed according to Li et al. (2017) and the manufacturer’s instructions (Clontech).

To make constructs for BiLC experiments, the ORFs of DEK40 and PBAC3 of maize were cloned into JW771 (NLUC) and JW772 (CLUC), respectively yielding DEK40-NLUC and PBAC3-CLUC constructs. Agrobacterium (strain GV3101) harboring the DEK40-NLUC and PBAC3-CLUC constructs was infiltrated into 3-week–old Nicotiana benthamiana leaves. After growing for 72 h under the condition of 16 h of light and 8 h of dark, leaves were injected with 0.8 mm of luciferin and the resulting luciferase signals were captured using a Tanon-5200 system (https://abclonal.com/products-tanon/tanon-5200ce-chemi-image-system/5200CE). Quantitative analysis was performed using the software ImageJ (Li et al., 2017). At least three independent experiments were performed for each group.

Proteasome Complexes Isolation and Blue Native-PAGE

The 26S proteasome complexes were purified from wild-type and dek40 kernels at 12, 18, and 24 DAP according to Book et al. (2010) and Li et al. (2015a). Immature seeds were frozen to liquid nitrogen temperatures, pulverized, and ground in 1.25 volumes of buffer A (50-mm Tris-HCl at pH 7.5, 10% [v/v] glycerol, 5-mm ATP, 5-mm MgCl2, 1-mm DTT, and 1-mg/mL creatine phosphokinase). After filtering through a PES Membrane (Thermo Fisher Scientific), the extract was clarified for 15 min at 12,000g. The supernatant was transferred to an ultracentrifuge tube and centrifuged at 35,500g for 20 min 4°C. The supernatant was subjected to FPLC using a Superdex 200 10/300GL column (GE Healthcare) equilibrated with buffer A and collecting 0.5-mL fractions. The 20 samples from the fraction collector were placed on ice as soon as the run was completed. The various proteasome complexes (9–18) of wild-type kernels at 18 DAP were separated by 12.5% SDS-PAGE and analyzed by immunoblotting using anti-DEK40, antiα3, and antiβ2 antibodies. For native gels, the mixture of various proteasome complexes (1–12, ≥ 670 kD) of wild-type and dek40 mutant kernels at 18 DAP were separated by 4.5% native PAGE at 4°C and analyzed by immunoblotting using antiβ2 antibody and anti-Rpt5 antibody, respectively. The electrophoresis was run at 15 mA until the loading dye migrated to the edge of the gel according to Dohmen et al. (2005).

Proteasome Activity Assay

The various proteasome complexes (1–12, ≥ 670 kD) of wild-type and dek40 mutant kernels at 12 DAP or 24 DAP were mixed. The mixed complexes were used for the proteasome activity assay according to the manufacturer’s instructions (CHEMICON Proteasome Activity Assay Kit, Cat. No. APT280; Merck Millipore). In brief, 10 μL of mixed complexes were added to the reaction buffer (final concentration: 25-mm HEPES at pH 7.5, 0.5-mm EDTA, 0.05% [v/v] NP-40 and 0.001% [w/v] SDS), and then fluorogenic peptide substrate Suc-LLVY-AMC (Merck Millipore) was added to a final concentration of 1 mM. The reaction (total volume of 100 μL) was conducted in a 96-well fluorometer plate for 2 h at 37°C or 45°C. Fluorescence data were collected at 360-nm excitation and 460-nm emission. For the inhibition assay, 25 μm of lactacysin was preincubated with proteasome extract for 15 min at room temperature, after which Suc-LLVY-AMC substrate was added. At least three independent experiments were performed for each group.

Ubiquitylome Analysis

The ubiquitylome experiments were performed with three independent biological replications. Each replication was pooled with 45 representative 15-DAP kernels from three individual dek40 and wild-type plants. Immunoaffinity purification of K-GG–modified peptides was performed using a PTMScan Ubiquitin Remnant Motif K-ε-GG Kit (Cell Signaling Technology). Ubiquitinated peptides were analyzed using the LC-MS/MS method according to the description in Figure 8A. The MS data were analyzed using the software MaxQuant v1.3.0.5 (https://www.maxquant.org/). MS data were searched against the UniProt Z. mays database. Significant differentially ubiquitinated peptides were identified as proteins with at least a 2.0-fold change in expression and P value < 0.05. The gene set enrichment analysis was performed according to Li et al. (2015b).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: ZmPBAC4 (Dek40), NP_001339840.1, GRMZM2G019538; S. bicolor (SbPAC4), KXG33006.1, SORBI_3003G239800; O. sativa (OsPAC4), BAT16164.1, Os12g0182800; Arabidopsis (AtPAC4), NP_564522.1, AT1G48170; Homo sapiens (HsPAC4), NP_001122064.1; H. sapiens (HsPAC3), NP_001127812.1; H. sapiens (HsPAC2), NP_064617.2; H. sapiens (HsPAC1), NP_003711.1; S. cerevisiae (ScPAC4), NP_015181.1; α-tubulin, GRMZM2G099167; α3, GRMZM2G130398; β2, GRMZM2G111566; heavy-chain binding protein, NM_001112423, GRMZM2G114793; PBAC3, NP_001142453.1, GRMZM2G028902.

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Ding Lu (Shanghai University) for maize transformation experiments, and Yanqing Ma (Shanghai University) for helping with proteasome complexes isolation.

Footnotes

1

This work was supported by the Natural Science Foundation of Shanghai (grant 17ZR1409400), the National Natural Foundation of China (grant nos. 31171559, 31771800, and 31370035), the Key Project of Shanghai Municipal Agricultural Commission (grant HNKGZ[2015]-6-1-2), and the Shanghai Pujiang Program (grant 17PJD014).

References

  1. Adams J. (2004) The proteasome: A suitable antineoplastic target. Nat Rev Cancer 4: 349–360 [DOI] [PubMed] [Google Scholar]
  2. Baumeister W, Walz J, Zühl F, Seemüller E (1998) The proteasome: Paradigm of a self-compartmentalizing protease. Cell 92: 367–380 [DOI] [PubMed] [Google Scholar]
  3. Bhattacharyya S, Yu H, Mim C, Matouschek A (2014) Regulated protein turnover: Snapshots of the proteasome in action. Nat Rev Mol Cell Biol 15: 122–133 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Book AJ, Gladman NP, Lee SS, Scalf M, Smith LM, Vierstra RD (2010) Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes. J Biol Chem 285: 25554–25569 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Budenholzer L, Cheng CL, Li Y, Hochstrasser M (2017) Proteasome structure and assembly. J Mol Biol 429: 3500–3524 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: Architecture and applications. BMC Bioinformatics 10: 421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collins GA, Goldberg AL (2017) The logic of the 26S proteasome. Cell 169: 792–806 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coux O, Tanaka K, Goldberg AL (1996) Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem 65: 801–847 [DOI] [PubMed] [Google Scholar]
  9. Demartino GN, Gillette TG (2007) Proteasomes: Machines for all reasons. Cell 129: 659–662 [DOI] [PubMed] [Google Scholar]
  10. Dohmen RJ, London MK, Glanemann C, Ramos PC (2005) Assays for proteasome assembly and maturation. Methods Mol Biol 301: 243–254 [DOI] [PubMed] [Google Scholar]
  11. Finley D. (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78: 477–513 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gerards WL, Enzlin J, Häner M, Hendriks IL, Aebi U, Bloemendal H, Boelens W (1997) The human alpha-type proteasomal subunit HsC8 forms a double ringlike structure, but does not assemble into proteasome-like particles with the beta-type subunits HsDelta or HsBPROS26. J Biol Chem 272: 10080–10086 [DOI] [PubMed] [Google Scholar]
  13. Gerards WL, de Jong WW, Bloemendal H, Boelens W (1998) The human proteasomal subunit HsC8 induces ring formation of other alpha-type subunits. J Mol Biol 275: 113–121 [DOI] [PubMed] [Google Scholar]
  14. Glickman MH. (2000) Getting in and out of the proteasome. Semin Cell Dev Biol 11: 149–158 [DOI] [PubMed] [Google Scholar]
  15. Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: Destruction for the sake of construction. Physiol Rev 82: 373–428 [DOI] [PubMed] [Google Scholar]
  16. Goldberg AL. (2007) Functions of the proteasome: From protein degradation and immune surveillance to cancer therapy. Biochem Soc Trans 35: 12–17 [DOI] [PubMed] [Google Scholar]
  17. Groll M, Bochtler M, Brandstetter H, Clausen T, Huber R (2005) Molecular machines for protein degradation. ChemBioChem 6: 222–256 [DOI] [PubMed] [Google Scholar]
  18. Guo J, Liu J, Wei Q, Wang R, Yang W, Ma Y, Chen G, Yu Y (2017) Proteomes and ubiquitylomes analysis reveals the involvement of ubiquitination in protein degradation in petunias. Plant Physiol 173: 668–687 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hirano Y, Hendil KB, Yashiroda H, Iemura S, Nagane R, Hioki Y, Natsume T, Tanaka K, Murata S (2005) A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes. Nature 437: 1381–1385 [DOI] [PubMed] [Google Scholar]
  20. Hirano Y, Hayashi H, Iemura S, Hendil KB, Niwa S, Kishimoto T, Kasahara M, Natsume T, Tanaka K, Murata S (2006) Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes. Mol Cell 24: 977–984 [DOI] [PubMed] [Google Scholar]
  21. Hirano Y, Kaneko T, Okamoto K, Bai M, Yashiroda H, Furuyama K, Kato K, Tanaka K, Murata S (2008) Dissecting beta-ring assembly pathway of the mammalian 20S proteasome. EMBO J 27: 2204–2213 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Horiguchi G, Ferjani A, Fujikura U, Tsukaya H (2006) Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana. J Plant Res 119: 37–42 [DOI] [PubMed] [Google Scholar]
  23. Hoyt MA, McDonough S, Pimpl SA, Scheel H, Hofmann K, Coffino P (2008) A genetic screen for Saccharomyces cerevisiae mutants affecting proteasome function, using a ubiquitin-independent substrate. Yeast 25: 199–217 [DOI] [PubMed] [Google Scholar]
  24. Kaneko T, Hamazaki J, Iemura S, Sasaki K, Furuyama K, Natsume T, Tanaka K, Murata S (2009) Assembly pathway of the Mammalian proteasome base subcomplex is mediated by multiple specific chaperones. Cell 137: 914–925 [DOI] [PubMed] [Google Scholar]
  25. Kim DY, Scalf M, Smith LM, Vierstra RD (2013) Advanced proteomic analyses yield a deep catalog of ubiquitylation targets in Arabidopsis. Plant Cell 25: 1523–1540 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33: 1870–1874 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kusmierczyk AR, Kunjappu MJ, Funakoshi M, Hochstrasser M (2008) A multimeric assembly factor controls the formation of alternative 20S proteasomes. Nat Struct Mol Biol 15: 237–244 [DOI] [PubMed] [Google Scholar]
  28. Kusmierczyk AR, Kunjappu MJ, Kim RY, Hochstrasser M (2011) A conserved 20S proteasome assembly factor requires a C-terminal HbYX motif for proteasomal precursor binding. Nat Struct Mol Biol 18: 622–629 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Le Tallec B, Barrault MB, Courbeyrette R, Guérois R, Marsolier-Kergoat MC, Peyroche A (2007) 20S proteasome assembly is orchestrated by two distinct pairs of chaperones in yeast and in mammals. Mol Cell 27: 660–674 [DOI] [PubMed] [Google Scholar]
  30. Li Q, Wang J, Ye J, Zheng X, Xiang X, Li C, Fu M, Wang Q, Zhang Z, Wu Y (2017) The maize imprinted gene Floury3 encodes a PLATZ protein required for tRNA and 5S rRNA transcription through interaction with RNA polymerase III. Plant Cell 29: 2661–2675 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Li XM, Chao DY, Wu Y, Huang X, Chen K, Cui LG, Su L, Ye WW, Chen H, Chen HC, et al. (2015b) Natural alleles of a proteasome α2 subunit gene contribute to thermotolerance and adaptation of African rice. Nat Genet 47: 827–833 [DOI] [PubMed] [Google Scholar]
  32. Li Y, Tomko RJ Jr., Hochstrasser M (2015a) Proteasomes: Isolation and activity assays. Curr Protoc Cell Biol 67: 1–20 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matias AC, Ramos PC, Dohmen RJ (2010) Chaperone-assisted assembly of the proteasome core particle. Biochem Soc Trans 38: 29–33 [DOI] [PubMed] [Google Scholar]
  34. Murata S, Udono H, Tanahashi N, Hamada N, Watanabe K, Adachi K, Yamano T, Yui K, Kobayashi N, Kasahara M, et al. (2001) Immunoproteasome assembly and antigen presentation in mice lacking both PA28α and PA28β. EMBO J 20: 5898–5907 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Murata S, Sasaki K, Kishimoto T, Niwa S, Hayashi H, Takahama Y, Tanaka K (2007) Regulation of CD8+ T cell development by thymus-specific proteasomes. Science 316: 1349–1353 [DOI] [PubMed] [Google Scholar]
  36. Murata S, Yashiroda H, Tanaka K (2009) Molecular mechanisms of proteasome assembly. Nat Rev Mol Cell Biol 10: 104–115 [DOI] [PubMed] [Google Scholar]
  37. Muratani M, Tansey WP (2003) How the ubiquitin-proteasome system controls transcription. Nat Rev Mol Cell Biol 4: 192–201 [DOI] [PubMed] [Google Scholar]
  38. Park S, Tian G, Roelofs J, Finley D (2010) Assembly manual for the proteasome regulatory particle: The first draft. Biochem Soc Trans 38: 6–13 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Qi W, Zhu J, Wu Q, Wang Q, Li X, Yao D, Jin Y, Wang G, Wang G, Song R (2016) Maize reas1 mutant stimulates ribosome use efficiency and triggers distinct transcriptional and translational responses. Plant Physiol 170: 971–988 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ramos PC, Dohmen RJ (2008) PACemakers of proteasome core particle assembly. Structure 16: 1296–1304 [DOI] [PubMed] [Google Scholar]
  41. Rosenzweig R, Glickman MH (2008) Chaperone-driven proteasome assembly. Biochem Soc Trans 36: 807–812 [DOI] [PubMed] [Google Scholar]
  42. Sahara K, Kogleck L, Yashiroda H, Murata S (2014) The mechanism for molecular assembly of the proteasome. Adv Biol Regul 54: 51–58 [DOI] [PubMed] [Google Scholar]
  43. Sasaki K, Hamazaki J, Koike M, Hirano Y, Komatsu M, Uchiyama Y, Tanaka K, Murata S (2010) PAC1 gene knockout reveals an essential role of chaperone-mediated 20S proteasome biogenesis and latent 20S proteasomes in cellular homeostasis. Mol Cell Biol 30: 3864–3874 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shibahara T, Kawasaki H, Hirano H (2004) Mass spectrometric analysis of expression of ATPase subunits encoded by duplicated genes in the 19S regulatory particle of rice 26S proteasome. Arch Biochem Biophys 421: 34–41 [DOI] [PubMed] [Google Scholar]
  45. Smalle J, Kurepa J, Yang P, Babiychuk E, Kushnir S, Durski A, Vierstra RD (2002) Cytokinin growth responses in Arabidopsis involve the 26S proteasome subunit RPN12. Plant Cell 14: 17–32 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tanaka K. (2009) The proteasome: Overview of structure and functions. Proc Jpn Acad, Ser B, Phys Biol Sci 85: 12–36 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Unno M, Mizushima T, Morimoto Y, Tomisugi Y, Tanaka K, Yasuoka N, Tsukihara T (2002) The structure of the mammalian 20S proteasome at 2.75 Å resolution. Structure 10: 609–618 [DOI] [PubMed] [Google Scholar]
  48. Wagner SA, Beli P, Weinert BT, Nielsen ML, Cox J, Mann M, Choudhary C (2011) A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol Cell Proteomics 10: 013284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang G, Sun X, Wang G, Wang F, Gao Q, Sun X, Tang Y, Chang C, Lai J, Zhu L, et al. (2011) Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm. Genetics 189: 1281–1295 [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  50. Wang G, Wang G, Zhang X, Wang F, Song R (2012) Isolation of high quality RNA from cereal seeds containing high levels of starch. Phytochem Anal 23: 159–163 [DOI] [PubMed] [Google Scholar]
  51. Wang G, Zhang J, Wang G, Fan X, Sun X, Qin H, Xu N, Zhong M, Qiao Z, Tang Y, et al. (2014) Proline responding1 plays a critical role in regulating general protein synthesis and the cell cycle in maize. Plant Cell 26: 2582–2600 [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  52. Williams-Carrier R, Stiffler N, Belcher S, Kroeger T, Stern DB, Monde RA, Coalter R, Barkan A (2010) Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy Mutator lines of maize. Plant J 63: 167–177 [DOI] [PubMed] [Google Scholar]
  53. Wolf DH, Hilt W (2004) The proteasome: A proteolytic nanomachine of cell regulation and waste disposal. Biochim Biophys Acta 1695: 19–31 [DOI] [PubMed] [Google Scholar]
  54. Xie X, Kang H, Liu W, Wang GL (2015) Comprehensive profiling of the rice ubiquitome reveals the significance of lysine ubiquitination in young leaves. J Proteome Res 14: 2017–2025 [DOI] [PubMed] [Google Scholar]
  55. Yang P, Fu H, Walker J, Papa CM, Smalle J, Ju YM, Vierstra RD (2004) Purification of the Arabidopsis 26 S proteasome: Biochemical and molecular analyses revealed the presence of multiple isoforms. J Biol Chem 279: 6401–6413 [DOI] [PubMed] [Google Scholar]
  56. Yashiroda H, Mizushima T, Okamoto K, Kameyama T, Hayashi H, Kishimoto T, Niwa S, Kasahara M, Kurimoto E, Sakata E, et al. (2008) Crystal structure of a chaperone complex that contributes to the assembly of yeast 20S proteasomes. Nat Struct Mol Biol 15: 228–236 [DOI] [PubMed] [Google Scholar]
  57. Zhong L, Belote JM (2007) The testis-specific proteasome subunit Prosα6T of D. melanogaster is required for individualization and nuclear maturation during spermatogenesis. Development 134: 3517–3525 [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES