Human SOD1 is secreted via a conventional secretion pathway in Saccharomyces cerevisiae.

Soluble proteins sorted through the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER) from the cytosol. However, a few proteins that lack a signal peptide are still translocated into the ER, such as SOD1. SOD1 is a causative gene of amyotrophic lateral sclerosis (ALS). A relationship has been suggested between the secretion of SOD1 and the pathogenesis of ALS; however, the transport mechanism of SOD1 remains unclear. We herein report that SOD1 was translocated into the ER lumen through the translocon Sec61 and was then secreted extracellularly. The present results indicate the potential of suppressing the secretion of SOD1 as a therapeutic target for ALS.

Golgi localization of glycosyltransferases requires Gpp74p in Schizosaccharomyces pombe.

The majority of Golgi glycosyltransferases are type II membrane proteins with a small cytosolic tail at their N-terminus. Several mechanisms for localizing these glycosyltransferases to the Golgi have been proposed. In Saccharomyces cerevisiae, the phosphatidylinositol-4-phosphate-binding protein ScVps74p interacts with the cytosolic tail of a Golgi glycosyltransferase and contributes to its localization. In this study, we investigated whether a similar mechanism functions in the fission yeast Schizosaccharomyces pombe. First, we identified gpp74+ (GPP34 domain-containing Vps74 homolog protein), a gene encoding the S. pombe homolog of S. cerevisiae Vps74p. Deletion of the gpp74+ gene resulted in the missorting of three Golgi glycosyltransferases, SpOch1p, SpMnn9p, and SpOmh1p, to vacuoles, but not SpAnp1p, indicating Gpp74p is required for targeting some glycosyltransferases to the Golgi apparatus. Gpp74p with an N-terminal GFP-tag localized to both the Golgi apparatus and the cytosol. Golgi localization of Gpp74p was dependent on the phosphatidylinositol 4-kinase SpPik1p. Site-directed mutagenesis of hydrophobic and basic amino acids in the cytosolic tails of SpOch1p and SpMnn9p resulted in their missorting to vacuoles, indicating these cytosolic N-terminal residues are important for localization in the Golgi. Unexpectedly, no prominent alternations in protein glycosylation were observed in S. pombe gpp74Δ cells, probably due to the residual Golgi localization of some SpOch1p and SpMnn9p in these cells. Collectively, these results demonstrate that both Gpp74p-dependent and Gpp74p-independent mechanisms are responsible for the Golgi localization of glycosyltransferases to the Golgi in S. pombe. KEY POINTS: • Gpp74p is involved in the localization of glycosyltransferases to the Golgi. • The cytosolic tails of glycosyltransferases are important for Golgi localization. • Gpp74p localizes to the Golgi in a SpPik1p-dependent manner.

The ER-associated protease Ste24 prevents N-terminal signal peptide-independent translocation into the endoplasmic reticulum in Saccharomyces cerevisiae.

Soluble proteins destined for the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER). The importance of N-terminal signal peptides for ER translocation has been extensively examined over the past few decades. However, in the budding yeast Saccharomyces cerevisiae, a few proteins devoid of a signal peptide are still translocated into the ER and then N-glycosyl-ated. Using signal peptide-truncated reporter proteins, here we report the detection of significant translocation of N-terminal signal peptide-truncated proteins in a yeast mutant strain (ste24Δ) that lacks the endopeptidase Ste24 at the ER membrane. Furthermore, several ER/cytosolic proteins, including Sec61, Sec66, and Sec72, were identified as being involved in the translocation process. On the basis of screening for 20 soluble proteins that may be N-glycosylated in the ER in the ste24Δ strain, we identified the transcription factor Rme1 as a protein that is partially N-glycosylated despite the lack of a signal peptide. These results clearly indicate that some proteins lacking a signal peptide can be translocated into the ER and that Ste24 typically suppresses this process.

Enrichment and characterization of a bacterial mixture capable of utilizing C-mannosyl tryptophan as a carbon source.

C-Mannosylation, a protein-modification found in various eukaryotes, involves the attachment of a single mannose molecule to selected tryptophan residues of proteins. Since C-mannosyl tryptophan (CMW) was detected in human urine, it is generally thought that CMW is not catabolized inside our body and instead is excreted via the urine. This paper reports enrichment of a bacterial consortium from soil that degrades CMW. The bacteria grew in minimal medium supplemented with CMW as the carbon source. Interestingly, even after successive clonal picks of individual colonies, several species were still present in each colony as revealed by 16S rRNA gene sequence analysis, indicating that a single species may not be responsible for this activity. A next generation sequencing (NGS) analysis was therefore carried out in order to determine which bacteria were responsible for the catabolism of CMW. It was found that a species of Sphingomonadaceae family, but not others, increased with simultaneous decrease of CMW in the media, suggesting that this species is most likely the one that is actively involved in the degradation of CMW.

Tissue-specific regulation of BMP signaling by Drosophila N-glycanase 1.

Mutations in the human N-glycanase 1 (NGLY1) cause a rare, multisystem congenital disorder with global developmental delay. However, the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known. Here we show that Drosophila Pngl encodes an N-glycanase and exhibits a high degree of functional conservation with human NGLY1. Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling. Pngl mutant larvae also exhibit a severe midgut clearance defect, which cannot be fully explained by impaired BMP signaling. Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development. Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers. Thus, our studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N-glycanase enzyme.

Identification of PNGase-dependent ERAD substrates in Saccharomyces cerevisiae.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a proteolytic pathway for handling misfolded or improperly assembled proteins that are synthesized in the ER. Cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans that are attached to ERAD substrates. While the critical roles of N-glycans in monitoring the folding status of carrier proteins in the ER lumen are relatively well understood, the physiological role of PNGase-mediated deglycosylation in the cytosol remained poorly understood. We report herein the identification of endogenous substrates for the cytoplasmic PNGase in Saccharomyces cerevisiae Using an isotope-coded glycosylation site-specific tagging (IGOT) method-based LC/MS analysis, 11 glycoproteins were specifically detected in the cytosol of PNGase-deletion cells (png1Δ). Among these molecules, at least five glycoproteins were clearly identified as ERAD substrates in vivo Moreover, four out of the five proteins were found to be either deglycosylated by PNGase in vivo or the overall degradation was delayed in a png1Δ mutant. Our results clearly indicate that the IGOT method promises to be a powerful tool for the identification of endogenous substrates for the cytoplasmic PNGase.

Endo-ß-N-acetylglucosaminidase forms N-GlcNAc protein aggregates during ER-associated degradation in Ngly1-defective cells.

The cytoplasmic peptide:N-glycanase (PNGase; Ngly1 in mice) is a deglycosylating enzyme involved in the endoplasmic reticulum (ER)-associated degradation (ERAD) process. The precise role of Ngly1 in the ERAD process, however, remains unclear in mammals. The findings reported herein, using mouse embryonic fibroblast (MEF) cells, that the ablation of Ngly1 causes dysregulation of the ERAD process. Interestingly, not only delayed degradation but also the deglycosylation of a misfolded glycoprotein was observed in Ngly1(-/-) MEF cells. The unconventional deglycosylation reaction was found to be catalyzed by the cytosolic endo-ß-N-acetylglucosaminidase (ENGase), generating aggregation-prone N-GlcNAc proteins. The ERAD dysregulation in cells lacking Ngly1 was restored by the additional knockout of ENGase gene. Thus, our study underscores the functional importance of Ngly1 in the ERAD process and provides a potential mechanism underlying the phenotypic consequences of a newly emerging genetic disorder caused by mutation of the human NGLY1 gene.

Vsl1p cooperates with Fsv1p for vacuolar protein transport and homotypic fusion in Schizosaccharomyces pombe.

Members of the SNARE protein family participate in the docking-fusion step of several intracellular vesicular transport events. Saccharomyces cerevisiae Vam7p was identified as a SNARE protein that acts in vacuolar protein transport and membrane fusion. However, in Schizosaccharomyces pombe, there have been no reports regarding the counterpart of Vam7p. Here, we found that, although the SPCC594.06c gene has low similarity to Vam7p, the product of SPCC594.06c has a PX domain and SNARE motif like Vam7p, and thus we designated the gene Sch. pombe vsl1(+) (Vam7-like protein 1). The vsl1Δ cells showed no obvious defect in vacuolar protein transport. However, cells of the vsl1Δ mutant with a deletion of fsv1(+), which encodes another SNARE protein, displayed extreme defects in vacuolar protein transport and vacuolar morphology. Vsl1p was localized to the vacuolar membrane and prevacuolar compartment, and its PX domain was essential for proper localization. Expression of the fusion protein GFP-Vsl1p was able to suppress ZnCl2 sensitivity and the vacuolar protein sorting defect in the fsv1Δ cells. Moreover, GFP-Vsl1p was mislocalized in a pep12Δ mutant and in cells overexpressing fsv1(+). Importantly, overexpression of Sac. cerevisiae VAM7 could suppress the sensitivity to ZnCl2 of vsl1Δ cells and the vacuolar morphology defect of vsl1Δfsv1Δ cells in Sch. pombe. Taken together, these data suggest that Vsl1p and Fsv1p are required for vacuolar protein transport and membrane fusion, and they function cooperatively with Pep12p in the same membrane-trafficking step.

Physiological and molecular functions of the cytosolic peptide:N-glycanase.

Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that acts on N-glycoproteins. A growing evidence exists to indicate that the cytosolic form of PNGase, which is ubiquitously distributed throughout eukaryotes, is not only implicated in the efficient degradation of misfolded glycoproteins destined for the proteasomal degradation but also in the generation of free oligosaccharides as the initial step in the non-lysosomal catabolism of N-glycans. This article summarizes the current state of our knowledge of the physiological and molecular functions of the cytosolic PNGase in a model organism, Saccharomyces cerevisiae, and also discusses the functional/structural diversities of this molecule within eukaryotes.

Cytoplasmic peptide:N-glycanase cleaves N-glycans on a carboxypeptidase Y mutant during ERAD in Saccharomyces cerevisiae.

BACKGROUND: Endoplasmic reticulum (ER)-associated degradation (ERAD) is a pathway by which misfolded or improperly assembled proteins in the ER are directed to degradation. The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans from misfolded glycoproteins during the ERAD process. The mutant form of yeast carboxypeptidase Y (CPY*) is an ERAD model substrate that has been extensively studied in yeast. While a delay in the degradation of CPY* in yeast cells lacking the cytoplasmic PNGase (Png1 in yeast) was evident, the in vivo action of PNGase on CPY* has not been detected. METHODS: We constructed new ERAD substrates derived from CPY*, bearing epitope tags at both N- and C-termini and examined the degradation intermediates observed in yeast cells with compromised proteasome activity. RESULTS: The occurrence of the PNGase-mediated deglycosylation of intact CPY* and its degradation intermediates was evident. A major endoproteolytic reaction on CPY* appears to occur between amino acid 400 and 404. CONCLUSIONS: The findings reported herein clearly indicate that PNGase indeed releases N-glycans from CPY* during the ERAD process in vivo. GENERAL SIGNIFICANCE: This report implies that the PNGase-mediated deglycosylation during the ERAD process may occur more abundantly than currently envisaged.

Endo-ß-N-acetylglucosamidases (ENGases) in the fungus Trichoderma atroviride: possible involvement of the filamentous fungi-specific cytosolic ENGase in the ERAD process.

N-Glycosylation is an important post-translational modification of proteins, which mainly occurs in the endoplasmic reticulum (ER). Glycoproteins that are unable to fold properly are exported to the cytosol for degradation by a cellular system called ER-associated degradation (ERAD). Once misfolded glycoproteins are exported to the cytosol, they are subjected to deglycosylation by peptide:N-glycanase (PNGase) to facilitate the efficient degradation of misfolded proteins by the proteasome. Interestingly, the ortholog of PNGase in some filamentous fungi was found to be an inactive deglycosylating enzyme. On the other hand, it has been shown that in filamentous fungi genomes, usually two different fungi-specific endo-ß-N-acetylglucosamidases (ENGases) can be found; one is predicted to be localized in the cytosol and the other to have a signal sequence, while the functional importance of these enzymes remains to be clarified. In this study the ENGases of the filamentous fungus Trichoderma atroviride was characterized. By heterologous expression of the ENGases Eng18A and Eng18B in Saccharomyces cerevisiae, it was found that both ENGases are active deglycosylating enzymes. Interestingly, only Eng18B was able to enhance the efficient degradation of the RTL protein, a PNGase-dependent ERAD substrate, implying the involvement of this enzyme in the ERAD process. These results indicate that T. atroviride Eng18B may deglycosylate misfolded glycoproteins, substituting the function of the cytoplasmic PNGase in the ERAD process.

A plant peptide: N-glycanase orthologue facilitates glycoprotein ER-associated degradation in yeast.

BACKGROUND: The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme involved in the ER-associated degradation (ERAD) process, while ERAD-independent activities are also reported. Previous biochemical analyses indicated that the cytoplasmic PNGase orthologue in Arabidopsis thaliana (AtPNG1) can function as not only PNGase but also transglutaminase, while its in vivo function remained unclarified. METHODS: AtPNG1 was expressed in Saccharomyces cerevisiae and its in vivo role on PNGase-dependent ERAD pathway was examined. RESULTS: AtPNG1 could facilitate the ERAD through its deglycosylation activity. Moreover, a catalytic mutant of AtPNG1 (AtPNG1(C251A)) was found to significantly impair the ERAD process. This result was found to be N-glycan-dependent, as the AtPNG(C251A) did not affect the stability of the non-glycosylated RTA∆ (ricin A chain non-toxic mutant). Tight interaction between AtPNG1(C251A) and the RTA∆ was confirmed by co-immunoprecipitation analysis. CONCLUSION: The plant PNGase facilitates ERAD through its deglycosylation activity, while the catalytic mutant of AtPNG1 impair glycoprotein ERAD by binding to N-glycans on the ERAD substrates. GENERAL SIGNIFICANCE: Our studies underscore the functional importance of a plant PNGase orthologue as a deglycosylating enzyme involved in the ERAD.

CUE domain-containing protein Vps901 is required for vacuolar protein transport in Schizosaccharomyces pombe.

The functions of two Schizosaccharomyces pombe Vps9-like genes, SPBC4F6.10/vps901(+) and SPBC29A10.11c/vps902(+), were characterized. Genomic sequence analysis predicted that Vps901p contains a VPS9 domain, whereas cDNA analyses revealed that Vps901p contains a CUE domain (coupling of ubiquitin to ER degradation) in its C-terminal region. Deletion of vps901(+) resulted in mis-sorting and secretion of S. pombe vacuolar carboxypeptidase Cpy1p, whereas deletion of vps902(+) had no effect, suggesting that only Vps901p functions in vacuolar protein transport in S. pombe. Deletion of vps901(+) further produced pleiotropic phenotypes, including vacuolar homotypic fusion and endocytosis defects. Heterologous expression of the budding yeast VPS9 gene corrected the CPY mis-sorting defect in vps901Δ cells. These findings suggest that the VPS9 domain of Vps901p is required for vacuolar protein trafficking in S. pombe.

Schizosaccharomyces pombe Pep12p is required for vacuolar protein transport and vacuolar homotypic fusion.

In eukaryotic cells, SNARE proteins are essential for intracellular vesicle trafficking. Several SNARE proteins are required for vacuolar protein transport and vacuolar biogenesis in Saccharomyces cerevisiae. Previously we demonstrated that one of the fission yeast SNARE proteins, Pep12p, is not required for vacuolar fusion process in Schizosaccharomyces pombe. We have re-examined the function of S. pombe Pep12p using the newly created pep12(+) deletion strain. Deletion of the fission yeast pep12(+) gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y-and various ion- and drug-sensitivities. GFP-Pep12 fusion protein is mostly localized at the vacuolar membrane and the prevacuolar compartment. The S. pombe pep12Δ mutation phenocopies that of vps33Δ, suggesting that both Pep12p and Vps33p act at the same membrane fusion step in S. pombe, and both mutations cause vacuolar deficiency.

Identification of an Htm1 (EDEM)-dependent, Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) pathway in Saccharomyces cerevisiae: application of a novel assay for glycoprotein ERAD.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related alpha-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.

The gld1+ gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe.

The budding yeast Saccharomyces cerevisiae is able to utilize glycerol as the sole carbon source via two pathways (glycerol 3-phosphate pathway and dihydroxyacetone [DHA] pathway). In contrast, the fission yeast Schizosaccharomyces pombe does not grow on media containing glycerol as the sole carbon source. However, in the presence of other carbon sources such as galactose and ethanol, S. pombe could assimilate glycerol and glycerol was preferentially utilized over ethanol and galactose. No equivalent of S. cerevisiae Gcy1/glycerol dehydrogenase has been identified in S. pombe. However, we identified a gene in S. pombe, SPAC13F5.03c (gld1 (+)), that is homologous to bacterial glycerol dehydrogenase. Deletion of gld1 caused a reduction in glycerol dehydrogenase activity and prevented glycerol assimilation. The gld1 Delta cells grew on 50 mM DHA as the sole carbon source, indicating that the glycerol dehydrogenase encoded by gld1 (+) is essential for glycerol assimilation in S. pombe. Strains of S. pombe deleted for dak1 (+) and dak2 (+) encoding DHA kinases could not grow on glycerol and showed sensitivity to a higher concentration of DHA. The dak1 Delta strain showed a more severe reduction of growth on glycerol and DHA than the dak2 Delta strain because the expression of dak1 (+) mRNA was higher than that of dak2 (+). In wild-type S. pombe, expression of the gld1 (+), dak1 (+), and dak2 (+) genes was repressed at a high concentration of glucose and was derepressed during glucose starvation. We found that gld1 (+) was regulated by glucose repression and that it was derepressed in scr1 Delta and tup12 Delta strains.

Mannosylinositol phosphorylceramide is a major sphingolipid component and is required for proper localization of plasma-membrane proteins in Schizosaccharomyces pombe.

In Saccharomyces cerevisiae, three classes of sphingolipids contain myo-inositol--inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide (MIPC) and mannosyldiinositol phosphorylceramide [M(IP)(2)C]. No fission yeast equivalent of Ipt1p, the inositolphosphotransferase that synthesizes M(IP)(2)C from MIPC, has been found in the Schizosaccharomyces pombe genome. Analysis of the sphingolipid composition of wild-type cells confirmed that MIPC is the terminal and most abundant complex sphingolipid in S. pombe. Three proteins (Sur1p, Csg2p and Csh1p) have been shown to be involved in the synthesis of MIPC from IPC in S. cerevisiae. The S. pombe genome has three genes (SPAC2F3.01, SPCC4F11.04c and SPAC17G8.11c) that are homologues of SUR1, termed imt1(+), imt2(+) and imt3(+), respectively. To determine whether these genes function in MIPC synthesis in S. pombe, single and multiple gene disruptants were constructed. Single imt disruptants were found to be viable. MIPC was not detected and IPC levels were increased in the triple disruptant, indicating that the three SUR1 homologues are involved in the synthesis of MIPC. GFP-tagged Imt1p, Imt2p and Imt3p localized to Golgi apparatus membranes. The MIPC-deficient mutant exhibited pleiotropic phenotypes, including defects in cellular and vacuolar morphology, and in localization of ergosterols. MIPC seemed to be required for endocytosis of a plasma-membrane-localized amino acid transporter, because sorting of the transporter from the plasma membrane to the vacuole was severely impaired in the MIPC-deficient mutant grown under nitrogen-limiting conditions. These results suggest that MIPC has multiple functions not only in the maintenance of cell and vacuole morphology but also in vesicular trafficking in fission yeast.

Platinum-catalyzed nucleophilic addition of vinylsilanes at the beta-position.

In the presence of catalytic amounts of PtCl(2) and metal iodides, beta-substituted vinylsilanes reacted with aldehydes at the beta-position to give allyl silyl ethers. The Pt-catalyzed addition to aromatic aldehydes proceeded efficiently in the presence of LiI. The combined use of PtCl(2) and MnI(2) was found to be effective in addition to aliphatic aldehydes.

Autophagy-deficient Schizosaccharomyces pombe mutants undergo partial sporulation during nitrogen starvation.

Autophagy is triggered when organisms sense radical environmental changes, including nutritional starvation. During autophagy, cytoplasmic components, including organelles, are enclosed within autophagosomes and are degraded upon lysosome-vacuole fusion. In this study, we show that processing of GFP-tagged Atg8 can serve as a marker for autophagy in the fission yeast Schizosaccharomyces pombe. Using this marker, 13 Atg homologues were also found to be required for autophagy in fission yeast. In budding yeast, autophagy-deficient mutants are known to be sterile, whereas in fission yeast we found that up to 30 % of autophagy-defective cells with amino acid auxotrophy were able to recover sporulation when an excess of required amino acids was supplied. Furthermore, we found that approximately 15 % of the autophagy-defective cells were also able to sporulate when a prototrophic strain was subjected to nitrogen starvation, which suggested that fission yeast may store sufficient intracellular nitrogen to allow partial sporulation under nitrogen-limiting conditions, although the majority of the nitrogen source is supplied by autophagy. Monitoring of the sporulation process revealed that the process was blocked non-specifically at various stages in the atg1Delta and atg12Delta mutants, possibly due to a shortage of amino acids. Taking advantage of this partial sporulation ability of fission yeast, we sought evidence for the existence of a recycling system for nitrogen sources during starvation.

Multiple functions of ergosterol in the fission yeast Schizosaccharomyces pombe.

Sterols are a major class of membrane lipids in eukaryotes. In Schizosaccharomyces pombe, sterol 24-C-methyltransferase (Erg6p), C-8 sterol isomerase (Erg2p), C-5 sterol desaturase (Erg31p, Erg32p), C-22 sterol desaturase (Erg5p) and C-24 (28) sterol reductase (Sts1p/Erg4p) have been predicted, but not yet determined, to catalyse a sequence of reactions from zymosterol to ergosterol. Disruption mutants of these genes were unable to synthesize ergosterol, and most were tolerant to the polyene drugs amphotericin B and nystatin. Disruption of erg31(+) or erg32(+) did not cause ergosterol deficiency or tolerance to polyene drugs, indicating that the two C-5 sterol desaturases have overlapping functions. GFP-tagged DRM (detergent-resistant membrane)-associated protein Pma1p localized to the plasma membrane in ergDelta mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in erg6Delta but not in other erg mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the ergDelta mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in Saccharomyces cerevisiae. Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins.