Vacuolar Hydrolases Research Articles

FgVAC1 is an Essential Gene Required for Golgi-to-Vacuole Transport and Fungal Development in Fusarium graminearum

Fusarium graminearum is an important plant pathogen that causes head blight in cereal crops such as wheat, barley, and rice worldwide. In this study, we identified and functionally characterized FgVAC1, an essential gene in F. graminearum that encodes a Rab5 effector involved in membrane tethering functions. The essentiality of FgVAC1 was confirmed through a conditional promoter replacement strategy using the zearalenone-inducible promoter (PZEAR). Cytological analyses revealed that FgVac1 colocalizes with FgRab51 on early endosomes and regulates the proper transport of the vacuolar hydrolase FgCpy1 to the vacuole. Suppression of FgVAC1 led to inhibited vegetative growth, reduced asexual and sexual reproduction, decreased deoxynivalenol (DON) biosynthesis, and diminished pathogenicity. Our findings highlight the significant role of FgVac1 in vacuolar protein sorting, fungal development, and plant infection in F. graminearum.

Two aminopeptidase I homologs convergently contribute to pathobiology of fungal entomopathogen Beauveria bassiana via divergent physiology-dependent autophagy pathways for vacuolar targeting

IntroductionIn yeast, the cytoplasm-to-vacuole targeting (Cvt) pathway acts as a biosynthetic autophagy-related process, in which vacuolar targeting of hydrolase is mediated by the machineries involved in the selective autophagy. However, the mechanistic insights into vacuolar targeting of hydrolases through the selective autophagy pathway still remain enigmatic in filamentous fungi. ObjectivesOur study aims to investigate the mechanisms involved in vacuolar targeting of hydrolases in filamentous fungi. MethodsThe filamentous entomopathogenic fungus Beauveria bassiana was used as a representative of filamentous fungi. We identified the homologs of yeast aminopeptidase I (Ape1) in B. bassiana by bioinformatic analyses and characterized their physiological roles by gene function analyses. Pathways for vacuolar targeting of hydrolases were investigated via molecular trafficking analyses. ResultsB. bassiana has two homologs of yeast aminopeptidase I (Ape1) which are designated as BbApe1A and BbApe1B. The two homologs of yeast Ape1 contribute to starvation tolerance, development, and virulence in B. bassiana. Significantly, BbNbr1 acts as a selective autophagy receptor to mediate the vacuolar targeting of the two Ape1 proteins, in which BbApe1B interacts with BbNbr1 also directly interacting with BbAtg8, and BbApe1A has an additional requirement of the scaffold protein BbAtg11 that interacts with BbNbr1 and BbAtg8. Protein processing occurs at both terminuses of BbApe1A and only at carboxyl terminus of BbApe1B, which is also dependent on the autophagy-related proteins. Together, the functions and translocation processes of the two Ape1 proteins are associated with autophagy in fungal lifecycle. ConclusionThis study reveals the functions and translocation processes for vacuolar hydrolases in the insect-pathogenic fungi and improves our understandings of the Nbr1-mediated vacuolar targeting pathway in the filamentous fungi.

Vacuolar Processing Enzymes in Plant Programmed Cell Death and Autophagy.

Vacuolar processing enzymes (VPEs) are plant cysteine proteases that are subjected to autoactivation in an acidic pH. It is presumed that VPEs, by activating other vacuolar hydrolases, are in control of tonoplast rupture during programmed cell death (PCD). Involvement of VPEs has been indicated in various types of plant PCD related to development, senescence, and environmental stress responses. Another pathway induced during such processes is autophagy, which leads to the degradation of cellular components and metabolite salvage, and it is presumed that VPEs may be involved in the degradation of autophagic bodies during plant autophagy. As both PCD and autophagy occur under similar conditions, research on the relationship between them is needed, and VPEs, as key vacuolar proteases, seem to be an important factor to consider. They may even constitute a potential point of crosstalk between cell death and autophagy in plant cells. This review describes new insights into the role of VPEs in plant PCD, with an emphasis on evidence and hypotheses on the interconnections between autophagy and cell death, and indicates several new research opportunities.

The entry of unclosed autophagosomes into vacuoles and its physiological relevance.

It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy.

Identifying Interaction Partners of Yeast Protein Disulfide Isomerases Using a Small Thiol-Reactive Cross-Linker: Implications for Secretory Pathway Proteostasis.

Protein disulfide isomerases (PDIs) function in forming the correct disulfide bonds in client proteins, thereby aiding the folding of proteins that enter the secretory pathway. Recently, several PDIs have been identified as targets of organic electrophiles, yet the client proteins of specific PDIs remain largely undefined. Here, we report that PDIs expressed in Saccharomyces cerevisiae are targets of divinyl sulfone (DVSF) and other thiol-reactive protein cross-linkers. Using DVSF, we identified the interaction partners that were cross-linked to Pdi1 and Eug1, finding that both proteins form cross-linked complexes with other PDIs, as well as vacuolar hydrolases, proteins involved in cell wall biosynthesis and maintenance, and many ER proteostasis factors involved ER stress signaling and ER-associated protein degradation (ERAD). The latter discovery prompted us to examine the effects of DVSF on ER quality control, where we found that DVSF inhibits the degradation of the ERAD substrate CPY*, in addition to covalently modifying Ire1 and blocking the activation of the unfolded protein response. Our results reveal that DVSF targets many proteins within the ER proteostasis network and suggest that these proteins may be suitable targets for covalent therapeutic development in the future.

ZZ domains keep cytosol to vacuole delivery whiZZing along.

Selective autophagy relies on adaptor proteins to bind and transport cargos (or substrates) to the lysosome or vacuole, yet the mechanisms for cargo recognition are not well understood. In this issue, Wang etal (2021) showed that in the fission yeast, Nbr1, a homolog of a mammalian selective autophagy adaptor, recognizes vacuolar hydrolases Ams1 and Ape4 through both versatile and cargo-specific interactions with the Nbr1 ZZ1 domain.

A LAPF/phafin1-like protein regulates TORC1 and lysosomal membrane permeabilization in response to endoplasmic reticulum membrane stress.

Lysosomal membrane permeabilization (LMP) is a poorly understood regulator of programmed cell death that involves leakage of luminal lysosomal or vacuolar hydrolases into the cytoplasm. In Saccharomyces cerevisiae, LMP can be induced by antifungals and endoplasmic reticulum stressors when calcineurin also has been inactivated. A genome-wide screen revealed Pib2, a relative of LAPF/phafin1 that regulates LMP in mammals, as a pro-LMP protein in yeast. Pib2 associated with vacuolar and endosomal limiting membranes in unstressed cells in a manner that depended on its FYVE domain and on phosphatidylinositol 3-phosphate (PI(3)P) biosynthesis. Genetic experiments suggest that Pib2 stimulates the activity of TORC1, a vacuole-associated protein kinase that is sensitive to rapamycin, in a pathway parallel to the Ragulator/EGO complex containing the GTPases Gtr1 and Gtr2. A hyperactivating mutation in the catalytic subunit of TORC1 restored LMP to the gtr1∆ and pib2∆ mutants and also prevented the synthetic lethality of the double mutants. These findings show novel roles of PI(3)P and Pib2 in the regulation of TORC1, which in turn promoted LMP and nonapoptotic death of stressed cells. Rapamycin prevented the death of the pathogenic yeast Candida albicans during exposure to fluconazole plus a calcineurin inhibitor, suggesting that TORC1 broadly promotes sensitivity to fungistats in yeasts.

The coordinated action of the MVB pathway and autophagy ensures cell survival during starvation

The degradation and recycling of cellular components is essential for cell growth and survival. Here we show how selective and non-selective lysosomal protein degradation pathways cooperate to ensure cell survival upon nutrient limitation. A quantitative analysis of starvation-induced proteome remodeling in yeast reveals comprehensive changes already in the first three hours. In this period, many different integral plasma membrane proteins undergo endocytosis and degradation in vacuoles via the multivesicular body (MVB) pathway. Their degradation becomes essential to maintain critical amino acids levels that uphold protein synthesis early during starvation. This promotes cellular adaptation, including the de novo synthesis of vacuolar hydrolases to boost the vacuolar catabolic activity. This order of events primes vacuoles for the efficient degradation of bulk cytoplasm via autophagy. Hence, a catabolic cascade including the coordinated action of the MVB pathway and autophagy is essential to enter quiescence to survive extended periods of nutrient limitation.

The yeast Saccharomyces cerevisiae: An overview of methods to study autophagy progression

Macroautophagy (hereafter autophagy) is a highly evolutionarily conserved process essential for sustaining cellular integrity, homeostasis, and survival. Most eukaryotic cells constitutively undergo autophagy at a low basal level. However, various stimuli, including starvation, organelle deterioration, stress, and pathogen infection, potently upregulate autophagy. The hallmark morphological feature of autophagy is the formation of the double-membrane vesicle known as the autophagosome. In yeast, flux through the pathway culminates in autophagosome–vacuole fusion, and the subsequent degradation of the resulting autophagic bodies and cargo by vacuolar hydrolases, followed by efflux of the breakdown products. Importantly, aberrant autophagy is associated with diverse human pathologies. Thus, there is a need for ongoing work in this area to further understand the cellular factors regulating this process. The field of autophagy research has grown exponentially in recent years, and although numerous model organisms are being used to investigate autophagy, the baker’s yeast Saccharomyces cerevisiae remains highly relevant, as there are significant and unique benefits to working with this organism. In this review, we will focus on the current methods available to evaluate and monitor autophagy in S. cerevisiae, which in several cases have also been subsequently exploited in higher eukaryotes.

Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting.

BackgroundBudding yeasts are often used to secrete foreign proteins, but the efficiency is variable. To identify roadblocks in the yeast secretory pathway, we used a monomeric superfolder GFP (msGFP) as a visual tracer in Saccharomyces cerevisiae and Pichia pastoris.ResultsOne roadblock for msGFP secretion is translocation into the ER. Foreign proteins are typically fused to the bipartite α-factor secretion signal, which consists of the signal sequence followed by the pro region. The α-factor signal sequence directs posttranslational translocation. For msGFP, posttranslational translocation is inefficient with the α-factor signal sequence alone but is stimulated by the pro region. This requirement for the pro region can be bypassed by using the Ost1 signal sequence, which has been shown to direct cotranslational translocation. A hybrid secretion signal consisting of the Ost1 signal sequence followed by the α-factor pro region drives efficient translocation followed by rapid ER export. A second roadblock for msGFP secretion in S. cerevisiae occurs during exit from the Golgi, when some of the msGFP molecules are diverted to the vacuole. Deletion of the sorting receptor Vps10 prevents vacuolar targeting of msGFP at the expense of missorting vacuolar hydrolases such as carboxypeptidase Y (CPY) to the culture medium. However, a truncation of Vps10 blocks vacuolar targeting of msGFP while permitting CPY to be sorted normally.ConclusionsWith budding yeasts, if the secretion or processing of a foreign protein is poor, we recommend two options. First, use the Ost1 signal sequence to achieve efficient entry into the secretory pathway while avoiding the processing issues associated with the α-factor pro region. Second, truncate Vps10 to suppress diversion to the vacuole. These insights obtained with msGFP highlight the value of applying cell biological methods to study yeast secretion.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0125-0) contains supplementary material, which is available to authorized users.

The Nutrient Stress-induced Small GTPase Rab5 Contributes to the Activation of Vesicle Trafficking and Vacuolar Activity

Rab family small GTPases regulate membrane trafficking by spatiotemporal recruitment of various effectors. However, it remains largely unclear how the expression and functions of Rab proteins are regulated in response to extracellular or intracellular stimuli. Here we show that Ypt53, one isoform of Rab5 in Saccharomyces cerevisiae, is up-regulated significantly under nutrient stress. Under non-stress conditions, Vps21, a constitutively expressed Rab5 isoform, is crucial to Golgi-vacuole trafficking and to vacuolar hydrolase activity. However, when cells are exposed to nutrient stress for an extended period of time, the up-regulated Ypt53 and the constitutive Vps21 function redundantly to maintain these activities, which, in turn, prevent the accumulation of reactive oxygen species and maintain mitochondrial respiration. Together, our results clarify the relative roles of these constitutive and nutrient stress-inducible Rab5 proteins that ensure adaptable vesicle trafficking and vacuolar hydrolase activity, thereby allowing cells to adapt to environmental changes.

Intracellular aspartic proteinase Apr1p of Candida albicans is required for morphological transition under nitrogen-limited conditions but not for macrophage killing.

Vacuolar hydrolases have been thoroughly characterized in Saccharomyces cerevisiae, but their homologues in the fungal pathogen Candida albicans have received less attention. The genes APR1 and CPY1 of C. albicans encode putative vacuolar aspartic proteinase and serine carboxypeptidase, respectively. We examined properties of apr1Δ and cpy1Δ mutants, showing that Cpy1p molecular species detected in cell lysates of apr1Δ and its parental strain did not differ in molar mass. Processing of Cpy1p precursor is apparently independent of Apr1p. This is in contrast to S. cerevisiae, where vacuolar aspartic proteinase Pep4p is known to participate in the activation of other vacuolar hydrolases including serine carboxypeptidase. We also found that both apr1Δ and cpy1Δ strains are able to form hyphae in nutrient-rich filamentation media. However, proline as a sole nitrogen source induced filamentation only in cpy1Δ and its parental strain, but not in apr1Δ. This indicates the importance of Apr1p for the morphological transition under nitrogen-limited conditions. Despite that, the ability of apr1Δ to kill murine macrophages was not reduced under the conditions tested.

Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting

Budding yeasts are often used to secrete foreign proteins, but the efficiency is variable. To identify roadblocks in the yeast secretory pathway, we used a monomeric superfolder GFP (msGFP) as a visual tracer in Saccharomyces cerevisiae and Pichia pastoris. One roadblock for msGFP secretion is translocation into the ER. Foreign proteins are typically fused to the bipartite α-factor secretion signal, which consists of the signal sequence followed by the pro region. The α-factor signal sequence directs posttranslational translocation. For msGFP, posttranslational translocation is inefficient with the α-factor signal sequence alone but is stimulated by the pro region. This requirement for the pro region can be bypassed by using the Ost1 signal sequence, which has been shown to direct cotranslational translocation. A hybrid secretion signal consisting of the Ost1 signal sequence followed by the α-factor pro region drives efficient translocation followed by rapid ER export. A second roadblock for msGFP secretion in S. cerevisiae occurs during exit from the Golgi, when some of the msGFP molecules are diverted to the vacuole. Deletion of the sorting receptor Vps10 prevents vacuolar targeting of msGFP at the expense of missorting vacuolar hydrolases such as carboxypeptidase Y (CPY) to the culture medium. However, a truncation of Vps10 blocks vacuolar targeting of msGFP while permitting CPY to be sorted normally. With budding yeasts, if the secretion or processing of a foreign protein is poor, we recommend two options. First, use the Ost1 signal sequence to achieve efficient entry into the secretory pathway while avoiding the processing issues associated with the α-factor pro region. Second, truncate Vps10 to suppress diversion to the vacuole. These insights obtained with msGFP highlight the value of applying cell biological methods to study yeast secretion.

Vesicular Trafficking and Stress Response Coupled to PI3K Inhibition by LY294002 as Revealed by Proteomic and Cell Biological Analysis

LY294002 is a synthetic quercetin-like compound, which, unlike wortmannin, is more specific inhibitor of phosphatidylinositol 3-kinase (PI3K). It inhibits endocytosis and vacuolar transport. We report here on the proteome-wide effects of LY294002 on Arabidopsis roots focusing on proteins involved in vesicular trafficking and stress response. At the subcellular level, LY294002 caused swelling and clustering of late endosomes leading to inhibition of vacuolar transport. At the proteome level, this compound caused changes in abundances of proteins categorized to 10 functional classes. Among proteins involved in vesicular trafficking, a small GTPase ARFA1f was more abundant, indicating its possible contribution to the aggregation and fusion of late endosomes triggered by LY294002. Our study provides new information on storage proteins and vacuolar hydrolases in vegetative tissues treated by LY294002. Vacuolar hydrolases were downregulated, while storage proteins were more abundant, suggesting that storage proteins were protected from degradation in swollen multivesicular bodies upon LY294002 treatment. Upregulation of 2S albumin was validated by immunoblotting and immunolabeling analyses. Our study also pointed to the control of antioxidant enzyme machinery by PI3K because LY294002 downregulated two isozymes of superoxide dismutase. This most likely occurred via PI3K-mediated downregulation of protein AtDJ1A. Finally, we discuss specificity differences of LY294002 and wortmannin against PI3K, which are reflected at the proteome level. Compared with wortmannin, LY294002 showed more narrow and perhaps also more specific effects on proteins, as suggested by gene ontology functional annotation.

Organelle acidification is important for localisation of vacuolar proteins in Saccharomyces cerevisiae

The acidic environments in the vacuole and other acidic organelles are important for many cellular processes in eukaryotic cells. In this study, we comprehensively investigated the roles of organelle acidification in vacuolar protein localisation in Saccharomyces cerevisiae. After repressing the acidification of acidic compartments by treatment with concanamycin A, a specific inhibitor of vacuolar H(+)-ATPase (V-ATPase), we examined the localisation of GFP-fused proteins that were predicted to localise in the vacuolar lumen or on the vacuolar membrane. Of the 73 proteins examined, 19 changed their localisation to the cytoplasmic region. Localisation changes were evaluated quantitatively using the image processing programme CalMorph. The delocalised proteins included vacuolar hydrolases, V-ATPase subunits, transporters and enzymes for membrane biogenesis, as well as proteins required for protein transport. These results suggest that many alterations in the localisation of vacuolar proteins occur after loss of the acidification of acidic compartments.

Structure of the Novel C-terminal Domain of Vacuolar Protein Sorting 30/Autophagy-related Protein 6 and Its Specific Role in Autophagy

Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

A Genome-Wide Immunodetection Screen in S. cerevisiae Uncovers Novel Genes Involved in Lysosomal Vacuole Function and Morphology

Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface – ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY). Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes – MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.

Routing Misfolded Proteins through the Multivesicular Body (MVB) Pathway Protects against Proteotoxicity

The secretory pathway maintains multiple quality control checkpoints. Initially, endoplasmic reticulum-associated degradation pathways monitor protein folding to retain and eliminate aberrant products. Despite its broad client range, some molecules escape detection and traffic to Golgi membranes. There, a poorly understood mechanism termed Golgi quality control routes aberrant proteins for lysosomal/vacuolar degradation. To better understand Golgi quality control, we examined the processing of the obligate substrate Wsc1p. Misfolded Wsc1p does not use routes of typical vacuolar membrane proteins. Instead, it partitions into intralumenal vesicles of the multivesicular body (MVB) pathway, mediated by the E3 ubiquitin ligase Rsp5p. Its subsequent transport to the vacuolar lumen is essential for complete molecule breakdown. Surprisingly, the transport mode plays a second crucial function in neutralizing potential substrate toxicity. Eliminating the MVB sorting signal diverted molecules to the vacuolar limiting membrane, resulting in the generation of toxic by-products. These data demonstrate a new role of the MVB pathway in protein quality control.

Aspartyl Aminopeptidase Is Imported from the Cytoplasm to the Vacuole by Selective Autophagy in Saccharomyces cerevisiae

Macroautophagy is a catabolic process by which cytosolic components are sequestered by double membrane vesicles called autophagosomes and sorted to the lysosomes/vacuoles to be degraded. Saccharomyces cerevisiae has adapted this mechanism for constitutive transport of the specific vacuolar hydrolases aminopeptidase I (Ape1) and α-mannosidase (Ams1); this process is called the cytoplasm to vacuole targeting (Cvt) pathway. The precursor form of Ape1 self-assembles into an aggregate-like structure in the cytosol that is then recognized by Atg19 in a propeptide-dependent manner. The interaction between Atg19 and autophagosome-forming machineries allows selective packaging of the Ape1-Atg19 complex by the autophagosome-like Cvt vesicle. Ams1 also forms oligomers and utilizes the Ape1 transport system by interacting with Atg19. Although the mechanism of selective transport of the Cvt cargoes has been well studied, it is unclear whether proteins other than Ape1 and Ams1 are transported via the Cvt pathway. We describe here that aspartyl aminopeptidase (Yhr113w/Ape4) is the third Cvt cargo, which is similar in primary structure and subunit organization to Ape1. Ape4 has no propeptide, and it does not self-assemble into aggregates. However, it binds to Atg19 in a site distinct from the Ape1- and Ams1-binding sites, allowing it to "piggyback" on the Ape1 transport system. In growing conditions, a small portion of Ape4 localizes in the vacuole, but its vacuolar transport is accelerated by nutrient starvation, and it stably resides in the vacuole lumen. We propose that the cytosolic Ape4 is redistributed to the vacuole when yeast cells need more active vacuolar degradation.

The Yeast vps Class E Mutants: The Beginning of the Molecular Genetic Analysis of Multivesicular Body Biogenesis

In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this “class E compartment” contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.