Autophagy In Saccharomyces Cerevisiae Research Articles

Sulfur starvation-induced autophagy in Saccharomyces cerevisiae involves SAM-dependent signaling and transcription activator Met4

Autophagy is a key lysosomal degradative mechanism allowing a prosurvival response to stresses, especially nutrient starvation. Here we investigate the mechanism of autophagy induction in response to sulfur starvation in Saccharomyces cerevisiae. We found that sulfur deprivation leads to rapid and widespread transcriptional induction of autophagy-related (ATG) genes in ways not seen under nitrogen starvation. This distinctive response depends mainly on the transcription activator of sulfur metabolism Met4. Consistently, Met4 is essential for autophagy under sulfur starvation. Depletion of either cysteine, methionine or SAM induces autophagy flux. However, only SAM depletion can trigger strong transcriptional induction of ATG genes and a fully functional autophagic response. Furthermore, combined inactivation of Met4 and Atg1 causes a dramatic decrease in cell survival under sulfur starvation, highlighting the interplay between sulfur metabolism and autophagy to maintain cell viability. Thus, we describe a pathway of sulfur starvation-induced autophagy depending on Met4 and involving SAM as signaling sulfur metabolite.

Atg1-dependent phosphorylation of Vps34 is required for dynamic regulation of the phagophore assembly site and autophagy in Saccharomyces cerevisiae

ABSTRACT Macroautophagy/autophagy is a key catabolic pathway in which double-membrane autophagosomes sequester various substrates destined for degradation, enabling cells to maintain homeostasis and survive under stressful conditions. Several autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS) and cooperatively function to generate autophagosomes. Vps34 is a class III phosphatidylinositol 3-kinase, and Atg14-containing Vps34 complex I plays essential roles in autophagosome formation. However, the regulatory mechanisms of yeast Vps34 complex I are still poorly understood. Here, we demonstrate that Atg1-dependent phosphorylation of Vps34 is required for robust autophagy activity in Saccharomyces cerevisiae. Following nitrogen starvation, Vps34 in complex I is selectively phosphorylated on multiple serine/threonine residues in its helical domain. This phosphorylation is important for full autophagy activation and cell survival. The absence of Atg1 or its kinase activity leads to complete loss of Vps34 phosphorylation in vivo, and Atg1 directly phosphorylates Vps34 in vitro, regardless of its complex association type. We also demonstrate that the localization of Vps34 complex I to the PAS provides a molecular basis for the complex I-specific phosphorylation of Vps34. This phosphorylation is required for the normal dynamics of Atg18 and Atg8 at the PAS. Together, our results reveal a novel regulatory mechanism of yeast Vps34 complex I and provide new insights into the Atg1-dependent dynamic regulation of the PAS. Abbreviations: ATG: autophagy-related; BARA: the repeated, autophagy-specific Co-IP: co-immunoprecipitation; GFP: green fluorescent protein; IP-MS: immunoprecipitation followed by tandem mass spectrometry; NTD: the N-terminal domain; PAS: phagophore assembly site; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns3K: phosphatidylinositol 3-kinase; SUR: structurally uncharacterized region; Vps34[KD]: Vps34D731N.

The autophagy protein BcAtg2 regulates growth, development and pathogenicity in the gray mold fungus Botrytis cinerea

Autophagy is an intracellular degradation process that facilitates material recycling to maintain cellular homeostasis in eukaryotes. Atg2 is a phospholipid transfer protein involved in cellular autophagy in Saccharomyces cerevisiae. To date, the role of Atg2 in growth, development and pathogenicity of the gray mold fungus Botrytis cinerea remains unknown. In this study, we identified and characterized an Atg2 ortholog, designated as BcAtg2, in B. cinerea. Deletion of BcATG2 resulted in a block of the autophagic process in B. cinerea. The ΔBcAtg2 mutant failed to produce sclerotia, and showed significant reduction in mycelial growth rate, formation of aerial mycelium and conidiation. In addition, the ΔBcAtg2 mutant lost the ability to form infection structures and cause symptom on host plants. All of these phenotypic changes in ΔBcAtg2 mutant were restored by targeted gene complementation. Moreover, BcAtg2 was demonstrated to physically interact with the phosphoinositide binding protein BcAtg18. Taken together, these results indicate that BcAtg2 plays an important role in vegetative growth, development and pathogenicity in B. cinerea.

Application of Metabolomics in the Study of Starvation-Induced Autophagy in Saccharomyces cerevisiae: A Scoping Review.

This scoping review is aimed at the application of the metabolomics platform to dissect key metabolites and their intermediates to observe the regulatory mechanisms of starvation-induced autophagy in Saccharomyces cerevisiae. Four research papers were shortlisted in this review following the inclusion and exclusion criteria. We observed a commonly shared pathway undertaken by S. cerevisiae under nutritional stress. Targeted and untargeted metabolomics was applied in either of these studies using varying platforms resulting in the annotation of several different observable metabolites. We saw a commonly shared pathway undertaken by S. cerevisiae under nutritional stress. Following nitrogen starvation, the concentration of cellular nucleosides was altered as a result of autophagic RNA degradation. Additionally, it is also found that autophagy replenishes amino acid pools to sustain macromolecule synthesis. Furthermore, in glucose starvation, nucleosides were broken down into carbonaceous metabolites that are being funneled into the non-oxidative pentose phosphate pathway. The ribose salvage allows for the survival of starved yeast. Moreover, acute glucose starvation showed autophagy to be involved in maintaining ATP/energy levels. We highlighted the practicality of metabolomics as a tool to better understand the underlying mechanisms involved to maintain homeostasis by recycling degradative products to ensure the survival of S. cerevisiae under starvation. The application of metabolomics has extended the scope of autophagy and provided newer intervention targets against cancer as well as neurodegenerative diseases in which autophagy is implicated.

The trehalose-6-phosphate phosphatase Tps2 regulates ATG8 transcription and autophagy in Saccharomyces cerevisiae

ABSTRACT Macroautophagy/autophagy is an important catabolic process for maintaining cellular homeostasis by adapting to various stress conditions. Autophagy is mediated by a double-membrane autophagosome, which sequesters a portion of cytoplasmic components for delivery to the vacuole. Several autophagy-related (ATG) genes play crucial roles in autophagosome formation. The induction of ATG genes must be tightly regulated to maintain a proper autophagic activity, but their regulatory mechanisms are still largely unknown. Here, we report that the trehalose-6-phosphate phosphatase Tps2 functions as a positive regulator of autophagy in Saccharomyces cerevisiae. Cellular trehalose levels do not affect autophagy regulation by Tps2. Loss of Tps2 leads to impaired autophagic flux and reduced ATG8 expre/ssion under nitrogen starvation. In tps2Δ cells, Ume6 is predominantly dephosphorylated and represses ATG8 transcription by binding to its promoter region. Tps2 regulates nuclear translocation and activation of Rim15 kinase, a negative regulator of Ume6, by causing the dissociation of Rim15 from the 14-3-3 proteins Bmh1/2 under nitrogen starvation, suggesting that Rim15 mediates the function of Tps2 as a positive regulator of ATG8 induction. Furthermore, Tps2 plays a crucial role in the dephosphorylation of Ser1061 and Thr1075 residues of Rim15, which is important for controlling the dissociation of Rim15 from Bmh1/2 under nitrogen starvation. Together, our results reveal the role of Tps2 as a positive regulator of autophagy and provide new insight into the regulatory mechanisms of ATG gene expression. Abbreviations: ATG: autophagy-related; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; DAPI: 4ʹ,6-diamidino-2-phenylindole; GFP: green fluorescent protein; PKA: protein kinase A; PtdIns3K: phosphatidylinositol 3-kinase; Rim15KI: kinase-inactive Rim15; Rim15-2A: Rim15S1061A,T1075A; TEM: transmission electron microscopy; TORC1: target of rapamycin complex 1

Impaired GCR1 transcription resulted in defective inositol levels, vacuolar structure and autophagy in Saccharomyces cerevisiae.

In yeast, the GCR1 transcription factor is involved in the regulation of glycolysis and its deletion exhibited growth defect, reduced inositol and phosphatidylinositol (PI) levels compared to WT cells. We observed a down regulation of the INO1 and PIS1 expression in gcr1∆ cells under both I- and I+ conditions and the over expression of GCR1 in gcr1∆ cells restored the growth, retrieved the expression of INO1, and PIS1 comparable to WT cells. In the gel shift assay, the Gcr1p binds to its consensus sequence CTTCC in PIS1 promoter and regulates its expression but not in INO1 transcription. The WT cells, under I- significantly reduced the expression of GCR1 and PIS1, but increased the expression of KCS1 and de-repressed INO1. The Kcs1p expression was reduced in gcr1∆ cells; this reduced INO1 expression resulting in abnormal vacuolar structure and reduced autophagy in Saccharomyces cerevisiae.

Induction of autophagy by phosphate starvation in an Atg11-dependent manner in Saccharomyces cerevisiae

Upon nutrient starvation, eukaryotic cells exploit autophagy to reconstruct cellular components. Although autophagy is induced by depletion of various nutrients such as nitrogen, carbon, amino acids, and sulfur in yeast, it was previously ambiguous whether phosphate depletion could trigger the induction of autophagy. Here, we showed that phosphate depletion induced autophagy in Saccharomyces cerevisiae, albeit to a lesser extent than nitrogen starvation. It is known that rapid inactivation of the target of rapamycin complex 1 (TORC1) signaling pathway contributes to Atg13 dephosphorylation, which is one of the cues for autophagy induction. We found that phosphate starvation caused Atg13 dephosphorylation with slower kinetics than nitrogen starvation, suggesting that poor autophagic activity during phosphate starvation was associated with slower inactivation of the TORC1 pathway. Phosphate starvation-induced autophagy requires Atg11, an adaptor protein for selective autophagy, but not its cargo recognition domain. These results suggested that Atg11 plays important roles in low-level nonselective autophagy.

Accessory Interaction Motifs in the Atg19 Cargo Receptor Enable Strong Binding to the Clustered Ubiquitin-related Atg8 Protein

Selective autophagy contributes to cellular homeostasis by delivering harmful material into the lysosomal system for degradation via vesicular intermediates referred to as autophagosomes. The cytoplasm-to-vacuole targeting pathway is a variant of selective autophagy in Saccharomyces cerevisiae during which hydrolases such as prApe1 are transported into the vacuole. In general, selectivity is achieved by autophagic cargo receptors that link the cargo to autophagosomal membranes because of their ability to simultaneously interact with the cargo and Atg8 proteins that coat the membrane. The Atg19 receptor contains multiple Atg8 interaction sites in its C terminus in addition to a canonical Atg8-interacting LC3-interacting region (LIR, with LC3 being a homolog of Atg8) motif, but their mode of interaction with Atg8 is unclear. Here we show, using a combination of NMR, microscopy-based interaction assays, and prApe1 processing experiments, that two additional sites interact with Atg8 in a LIR-like and thus mutually exclusive manner. We term these motifs accessory LIR motifs because their affinities are lower than that of the canonical LIR motif. Thus, one Atg19 molecule has the ability to interact with multiple Atg8 proteins simultaneously, resulting in a high-avidity interaction that may confer specific binding to the Atg8-coated autophagosomal membrane on which Atg8 is concentrated.

The fungicidal activity of amphotericin B requires autophagy-dependent targeting to the vacuole under a nutrient-starved condition in Saccharomyces cerevisiae

In this study, we demonstrated that in distilled water, a nutrient-starved condition that elicits autophagy in Saccharomyces cerevisiae, an array of autophagy-deficient mutants are resistant to the fungicidal effects of amphotericin B. In addition, we found that a dansyl-labelled derivative of the antibiotic colocalized with disintegrated vacuoles throughout the cytoplasm in the amphotericin B-sensitive parental strain suspended in distilled water. In contrast, the dansyl-labelled derivative was not internalized in the Δatg18 strain, which is deficient in the formation of autophagosomes, a key early step in autophagy. However, the derivative accumulated without significant toxicity in structurally intact vacuoles in the Δvma1 mutant, which is deficient in the degradation of autophagic bodies, the final stage in autophagy. Our data support the idea that amphotericin B can utilize autophagy-dependent trafficking into the intra-vacuolar lumen, where it interacts with the luminal leaf of the membrane to cause structurally catastrophic effects.

Ole1, fatty acid desaturase, is required for Atg9 delivery and isolation membrane expansion during autophagy in Saccharomyces cerevisiae.

ABSTRACTMacroautophagy, a major degradation pathway of cytoplasmic components, is carried out through formation of a double-membrane structure, the autophagosome. Although the involvement of specific lipid species in the formation process remains largely obscure, we recently showed that mono-unsaturated fatty acids (MUFA) generated by stearoyl-CoA desaturase 1 (SCD1) are required for autophagosome formation in mammalian cells. To obtain further insight into the role of MUFA in autophagy, in this study we analyzed the autophagic phenotypes of the yeast mutant of OLE1, an orthologue of SCD1. Δole1 cells were defective in nitrogen starvation-induced autophagy, and the Cvt pathway, when oleic acid was not supplied. Defects in elongation of the isolation membrane led to a defect in autophagosome formation. In the absence of Ole1, the transmembrane protein Atg9 was not able to reach the pre-autophagosomal structure (PAS), the site of autophagosome formation. Thus, autophagosome formation requires Ole1 during the delivery of Atg9 to the PAS/autophagosome from its cellular reservoir.

Bidirectional regulation between TORC1 and autophagy in Saccharomyces cerevisiae

It has been reported in various model organisms that autophagy and the target of rapamycin complex 1 (TORC1) signaling are strongly involved in eukaryotic cell aging and decreasing TORC1 activity extends longevity by an autophagy-dependent mechanism. Thus, to expand our knowledge of the regulation of eukaryotic cell aging, it is important to understand the relationship between TORC1 signaling and autophagy. Many researchers have shown that TORC1 represses autophagy under normal growth conditions, and TORC1 inactivation contributes to the upregulation of autophagy. However, it is poorly understood how autophagy is regulated or terminated when starvation is prolonged. Here, we report that bidirectional regulation between autophagy and TORC1 exists in the yeast Saccharomyces cerevisiae. We show that mutant cells with weak TORC1 activity maintain autophagy longer than wild-type cells, and TORC1 is partially reactivated under ongoing nitrogen starvation by an autophagy-dependent mechanism. In addition, we found that Atg13 is gradually rephosphorylated during prolonged nitrogen starvation, and the kinase activity of Atg1 is required for Atg13 rephosphorylation. Our data suggest that TORC1 can be substantially, if not fully, reactivated in an autophagy-dependent manner under ongoing starvation, and that partially reactivated TORC1 eventually plays a role in the attenuation of autophagy.

Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae

In eukaryotes, autophagy is a conserved protein degradation system that degrades cytoplasmic components by encompassing them with double-membrane structures, called autophagosomes, and delivering them to the lytic compartments of vacuoles/lysosomes. Certain Atg proteins are known to be involved in autophagy, yet the identity and function of lipid molecules involved remain largely unknown. We investigated the involvement of sphingolipids in autophagy using Saccharomyces cerevisiae. Inhibiting synthesis of the simplest complex sphingolipid, inositol phosphorylceramide (IPC), resulted in reduced autophagic activities. Similar results were obtained using myriocin, an inhibitor of the first step in sphingolipid synthesis. Our results indicate that sphingolipids, especially IPC, are required for autophagy. Inhibition of sphingolipid synthesis had no effect on formation of Atg12-Atg5 or Atg8-phosphatidylethanolamine conjugates, on maturation of vacuolar proteases, or on formation of the pre-autophagosomal structure (PAS). These results suggest that sphingolipids are not involved in the cellular signaling that leads to formation of the PAS, but may be involved in the process of autophagosome formation.

Mitochondria regulate autophagy by conserved signalling pathways

Autophagy is a conserved degradative process that is crucial for cellular homeostasis and cellular quality control via the selective removal of subcellular structures such as mitochondria. We demonstrate that a regulatory link exists between mitochondrial function and autophagy in Saccharomyces cerevisiae. During amino-acid starvation, the autophagic response consists of two independent regulatory arms-autophagy gene induction and autophagic flux-and our analysis indicates that mitochondrial respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1-Atg13 kinase complex to the pre-autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors-mitochondrial dysfunction and autophagy inhibition-for ageing, cancerogenesis, and neurodegeneration.

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.

Positive or Negative Roles of Different Cyclin-Dependent Kinase Pho85-Cyclin Complexes Orchestrate Induction of Autophagy in Saccharomyces cerevisiae

As a major intracellular degradation pathway, autophagy is tightly regulated to prevent cellular dysfunction in all eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 is a major regulator of autophagy. Several other nutrient-sensory kinases also play critical roles to precisely modulate autophagy; however, the network of regulatory mechanisms remains largely elusive. We used genetic analyses to elucidate the mechanism by which the stress-responsive, cyclin-dependent kinase Pho85 and its corresponding cyclin complexes antagonistically modulate autophagy in Saccharomyces cerevisiae. When complexed with cyclins Pho80 and Pcl5, Pho85 negatively regulates autophagy through downregulating the protein kinase Rim15 and the transcription factors Pho4 and Gcn4. The cyclins Clg1, Pcl1, and Pho80, in concert with Pho85, positively regulate autophagy through promoting the degradation of Sic1, a negative regulator of autophagy that targets Rim15. Our results suggest a model in which Pho85 and its cyclin complexes have opposing roles in autophagy regulation.

MgAtg9 trafficking in Magnaporthe oryzae

Autophagy is a vacuolar/lysosomal cytoplasmic recycling system in eukaryotic cells. ScATG9 is indispensable for autophagy in Saccharomyces cerevisiae. Here, we deleted MgATG9, the orthologue of ScATG9, via targeted gene replacement in the phytopathogenic filamentous fungus Magnaporthe oryzae, and then analyzed the cellular distribution pattern of EGFP-MgAtg9 in the Mgatg9∆ mutant. We detected an expression profile of multiple green dots in the conidial cell inoculated in rich media and in the appressoria differentiated from the conidia in H2O. Concurrent with the punctation, we found some fluorescent signals localized on the central vacuole of the submerged hyphae from the conidia cultured in rich media. Next, we introduced DsRed2-MgAtg8 into the Mgatg9∆ mutant expressing EGFP-MgAtg9 and observed partial overlap at multiple sites in the conidial cell, reminiscent of that in the mammalian system. Our findings further led to the postulation that the multiple sites where the two fusions colocalized tend to merge as a central structure in the conidial cell. Finally, we tested the expression of EGFP-MgAtg9 in null mutants of MgATG1, 2, 13 and 18, respectively. We speculate that MgAtg1, 2 and 18, but not MgAtg13, is required for MgAtg9 cycling through the multiple colocalization sites to its storage pools in the conidial cell of M. oryzae, and fusion of these colocalization sites into a central structure could be governed through other unidentified mechanisms.

Characterization of the Atg17–Atg29–Atg31 complex specifically required for starvation-induced autophagy in Saccharomyces cerevisiae

Nutrient starvation induces autophagy to degrade cytoplasmic materials in the vacuole/lysosomes. In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization. Here, we show that these proteins constitutively form an Atg17–Atg29–Atg31 ternary complex, in which phosphorylated Atg31 is included. Reconstitution analysis of the ternary complex in E. coli indicates that the three proteins are included in equimolar amounts in the complex. The molecular mass of a monomeric Atg17–Atg29–Atg31 complex is calculated at 97 kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198 kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation.

Perturbation of intracellular acyl-CoA metabolism induces the unfolded protein response pathway and autophagy in Saccharomyces cerevisiae

Perturbation of intracellular acyl-CoA metabolism induces the unfolded protein response pathway and autophagy in Saccharomyces cerevisiae

Interrelationships among Atg proteins during autophagy in Saccharomyces cerevisiae.

Macroautophagy is a bulk degradation process induced by nutrient starvation in eukaryotic cells. In Saccharomyces cerevisiae, 16 ATG genes are essential for autophagosome formation. Recently, we demonstrated that these ATG genes can be classified into three groups on the basis of localization of GFP-Atg8p/Aut7p and Atg5p/Apg5p-GFP under nutrient-rich conditions. In this study, we analysed the intracellular localization of GFP-Atg8p, an autophagosome marker, in atg mutants under autophagy-inducing conditions; the localization of GFP-Atg8p exhibited several distinct patterns. Subsequent analyses revealed epistatic interrelationships among Atg proteins during the process of autophagosome formation.

Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein.

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(DeltaATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein. This conjugate was not observed in cells expressing Gsa7(DeltaATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.