Global analysis of the glycoproteome in Saccharomyces cerevisiae reveals new roles for protein glycosylation in eukaryotes
2009; Springer Nature; Volume: 5; Issue: 1 Linguagem: Inglês
10.1038/msb.2009.64
ISSN1744-4292
AutoresLi Kung, Sheng‐ce Tao, Jiang Qian, Michael G. Smith, M Snyder, Heng Zhu,
Tópico(s)Studies on Chitinases and Chitosanases
ResumoArticle15 September 2009Open Access Global analysis of the glycoproteome in Saccharomyces cerevisiae reveals new roles for protein glycosylation in eukaryotes Li A Kung Li A Kung Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USAPresent address: Helicos BioSciences, Building 700 3rd Floor, One Kendall Square, Cambridge, MA 02139, USA Search for more papers by this author Sheng-Ce Tao Sheng-Ce Tao Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA The High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University, Shanghai, ChinaPresent address: Room 126, Wenxuan Building of Medicine, 800 Dongchuan Rd, Shanghai 200240, China Search for more papers by this author Jiang Qian Jiang Qian Department of Ophthalmology, Johns Hopkins University, Baltimore, MD, USA Search for more papers by this author Michael G Smith Michael G Smith Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA Search for more papers by this author Michael Snyder Corresponding Author Michael Snyder Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USAPresent address: Department of Genetics, Stanford University, Stanford, CA 94305, USA Search for more papers by this author Heng Zhu Corresponding Author Heng Zhu Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA The High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Li A Kung Li A Kung Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USAPresent address: Helicos BioSciences, Building 700 3rd Floor, One Kendall Square, Cambridge, MA 02139, USA Search for more papers by this author Sheng-Ce Tao Sheng-Ce Tao Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA The High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University, Shanghai, ChinaPresent address: Room 126, Wenxuan Building of Medicine, 800 Dongchuan Rd, Shanghai 200240, China Search for more papers by this author Jiang Qian Jiang Qian Department of Ophthalmology, Johns Hopkins University, Baltimore, MD, USA Search for more papers by this author Michael G Smith Michael G Smith Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA Search for more papers by this author Michael Snyder Corresponding Author Michael Snyder Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USAPresent address: Department of Genetics, Stanford University, Stanford, CA 94305, USA Search for more papers by this author Heng Zhu Corresponding Author Heng Zhu Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA The High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA Search for more papers by this author Author Information Li A Kung1,‡, Sheng-Ce Tao2,3,4,‡, Jiang Qian5, Michael G Smith1, Michael Snyder 1 and Heng Zhu 2,3 1Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA 2Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA 3The High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA 4Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University, Shanghai, China 5Department of Ophthalmology, Johns Hopkins University, Baltimore, MD, USA ‡These authors contributed equally to this work *Corresponding authors. Department of Molecular, Cellular, and Developmental Biology, Yale University, PO Box 208103, New Haven, CT 06620-8103, USA. Tel.: +1 203 432 6139; Fax: +1 203 432 3597; E-mail: [email protected] of Pharmacology and Molecular Sciences and the HiT Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Tel.: +1 410 502 0878; Fax: +1 410 502 1872; E-mail: [email protected] Molecular Systems Biology (2009)5:308https://doi.org/10.1038/msb.2009.64 Present address: Helicos BioSciences, Building 700 3rd Floor, One Kendall Square, Cambridge, MA 02139, USA Present address: Room 126, Wenxuan Building of Medicine, 800 Dongchuan Rd, Shanghai 200240, China PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To further understand the roles of protein glycosylation in eukaryotes, we globally identified glycan-containing proteins in yeast. A fluorescent lectin binding assay was developed and used to screen protein microarrays containing over 5000 proteins purified from yeast. A total of 534 yeast proteins were identified that bound either Concanavalin A (ConA) or Wheat-Germ Agglutinin (WGA); 406 of them were novel. Among the novel glycoproteins, 45 were validated by mobility shift upon treatment with EndoH and PNGase F, thereby extending the number of validated yeast glycoproteins to 350. In addition to many components of the secretory pathway, we identified other types of proteins, such as transcription factors and mitochondrial proteins. To further explore the role of glycosylation in mitochondrial function, the localization of four mitochondrial proteins was examined in the presence and absence of tunicamycin, an inhibitor of N-linked protein glycosylation. For two proteins, localization to the mitochondria is diminished upon tunicamycin treatment, indicating that protein glycosylation is important for protein function. Overall, our studies greatly extend our understanding of protein glycosylation in eukaryotes through the cataloguing of glycoproteins, and describe a novel role for protein glycosylation in mitochondrial protein function and localization. Synopsis Protein glycosylation is ubiquitous in eukaryotes and has been implicated in a wide variety of biochemical and cellular processes, including protein folding, maintenance of cell structure, receptor–ligand interactions and cell signalling, cell–cell recognition, and defence (Helenius and Aebi, 2004; Dube and Bertozzi, 2005). In spite of its importance, both the number and different types of proteins that are glycosylated are not known, and thus it is likely that the full range of biochemical and cellular functions is not understood. Thus, systematic studies would be useful to learn more about the roles of glycosylation. We set out to globally identify glycoproteins in S. cerevisiae by probing the yeast proteome chips with lectins that recognize GlcNAc or mannose. To achieve this, we developed a glycan competition assay that allowed for sensitive detection of specific glycans by circumventing the non-specific binding of ConA and WGA to the surfaces and 'sticky' protein spots while simultaneously maintaining strong signals. Also, to improve the coverage, both the two types of proteome microarray with either C-terminal tagged or N-terminal tagged protein were used in this assay (Figure 1). As a result, we greatly extended the number of known glycoproteins in yeast and identified several glycoproteins that localize to the mitochondrion. To further explore the function of glycosylation to these mitochondrial proteins, treatment of cells with an inhibitor of protein glycosylation disrupted the localization of two mitochondrial proteins were carried out (Ydr065wp and Lpe10p; Figure 6). In addition to providing a more comprehensive understanding of protein glycosylation in general, this study thus defines new roles for protein glycosylation in mitochondrial protein function and localization. Overall, our studies greatly extend our understanding of protein glycosylation in eukaryotes through cataloguing of glycoproteins, and describe a novel role for protein glycosylation in mitochondrial protein function and localization. Further definition and characterization of the glycome of yeast and other eukaryotes is expected to reveal additional novel roles or protein glycosylation in eukaryotes. Introduction Protein glycosylation is ubiquitous in eukaryotes and has been implicated in a wide variety of biochemical and cellular processes, including protein folding, maintenance of cell structure, receptor–ligand interactions and cell signalling, cell–cell recognition, and defence (Helenius and Aebi, 2004; Dube and Bertozzi, 2005). In spite of its importance, both the number and different types of proteins that are glycosylated are not known, and thus it is likely that the full range of biochemical and cellular functions is not understood. Two major types of protein glycosylation have been identified thus far: N- and O-linked. N-linked glycans are attached to asparagine residues and O-linked glycans are connected to serine or threonine residues. The local motif required for attachment is known only for N-linked glycans: N-X-S/T, where the point of attachment is upstream of either a serine or threonine, and the intervening amino acid cannot be proline (Gavel and von Heijne, 1990). A variety of different glycans are often attached to the modified proteins by glycotransferases in a series of processing steps. The degree of processing can be heterogeneous, resulting in a complex mixture of isoforms for a particular protein. N-linked glycosylation has been primarily associated with secretion, and most N-linked glycoproteins reside in compartments associated with this pathway. Glycan attachment is initiated in the endoplasmic reticulum (ER), where the nascent polypeptide chains are modified with an N-linked oligosaccharide at the appropriate site (Nilsson et al, 2003). The glycans on the glycoproteins are processed through the Golgi, and many proteins ultimately are shuttled to the plasma membrane or secreted. O-linked glycosylated proteins can be associated with membranes, in the cytosol, or within the nucleus (Cole and Hart, 1999; Wells and Hart, 2003). Few studies have investigated the role of protein glycosylation in mitochondrial function. Glycosyltransferase activity has been found in both the inner and outer membranes of mouse liver mitochondrion, although modified peptide chains were not identified (Levrat et al, 1989, 1990). In Saccharomyces cerevisiae, a hypomorphic allele of ALG7, encoding an enzyme involved in the first N-linked glycan addition, caused mitochondrial dysfunction (Mendelsohn et al, 2005). A previous study demonstrated that a member of the ubiquinone complex in the mitochondrial inner membrane from rat liver tissue is mannosylated and is transported from the ER to the mitochondria (Chandra et al, 1998). To our knowledge, this is the only mammalian mitochondrial glycoprotein that has been identified. To learn more about the roles of glycosylation, systematic studies would be useful. To date, a limited number of studies have been performed. Computational methods based on motif analysis are not sufficient to predict modified proteins. For example, a perusal of the S. cerevisiae proteome sequence indicates that the limited motif for N-linked glycosylation occurs ∼4.5 times per protein on average, yet most proteins are probably not glycosylated. Using covalent coupling to hydrazide resin, Zhang and Abersold purified glycoproteins from cell lysates, followed by mass spectrometry to identify hundreds of novel N-linked glycoproteins in mammalian cells (Kaji et al, 2003; Zhang et al, 2003; Tian et al, 2007). However, these studies do not identify low-abundance proteins. Yeast is an ideal system for studying protein glycosylation (Kukuruzinska et al, 1987). Twenty to 50% percent of the yeast proteins are estimated to be glycosylated (Apweiler et al, 1999), and 175 N- and O-linked proteins (3% of total proteins) have been previously identified through studies of individual proteins (Costanzo et al, 2000; Csank et al, 2002). Two glycans are known to be present on yeast proteins; N-linked chains have N-acetylglucosamine (GlcNAc) and mannose, whereas O-linked chains have only mannose (Gemmill and Trimble, 1999). Many of the glycotransferases responsible for adding and trimming carbohydrates in yeast are known (Herscovics, 1999). Finally, proteomic and genomic tools are readily available for studying protein glycosylation. In particular, two versions of yeast proteome arrays (Kung and Snyder, 2006), which contain most yeast proteins with either N- or C-terminal tags spotted at high density onto microscope slides, are useful for surveying protein glycosylation. Gelperin et al (2005) performed a preliminary systematic analysis of protein glycosylation in yeast by probing a proteome chip containing 5573 C-terminally tagged fusion proteins purified from yeast with an antibody raised against the glycan backbone of yeast glycoproteins. This study increased the number of N-linked glycoproteins known in S. cerevisiae from 106 to 231, and the total number of known glycoproteins to 305. The sensitivity of that study was limited by the antibody and only N-linked proteins could be identified. In this study, we have more comprehensively examined both the number and types of proteins that are glycosylated by developing and employing an assay using lectins to probe yeast proteome microarrays. Plant and fungal lectins have long been used as a tool to identify glycans on proteins (Chrispeels and Raikhel, 1991). Among the hundreds of known lectins, some (including Concanavalin A (ConA) and Wheat-Germ Agglutinin (WGA)) have been scrutinized for their glycan-binding spectra and affinities using glycan microarrays, thereby allowing specific detection of different glycoproteins (Hirabayashi, 2004). Using our approach, we greatly extended the number of known glycoproteins in yeast and identified several glycoproteins that localize to the mitochondrion. Treatment of cells with an inhibitor of protein glycosylation disrupted localization of two mitochondrial proteins (Gregan et al, 2001; Huh et al, 2003; Reinders et al, 2006). In addition to providing a more comprehensive understanding of protein glycosylation in general, this study thus defines new roles for protein glycosylation in mitochondrial protein function and localization. Results Development of a glycan-dependent lectin glycoprotein screen using proteome chips We set out to globally identify glycoproteins in S. cerevisiae by probing the yeast proteome chips with lectins that recognize GlcNAc or mannose. Two lectins were used, ConA, which recognizes mannose residues, and WGA, which recognizes GlcNAc. As shown by the glycan microarray experiments from the Consortium for Functional Glycomics, the selectivity of these lectins for their preferred glycans is much higher than for the other related glycans (http://www.functionalglycomics.org/glycomics/publicdata/primaryscreen.jsp). The lectins were used to screen two types of chips: (1) a chip containing ∼5800 proteins in which the proteins were fused at their N-termini to GST; (Zhu et al, 2001) and (2) an array containing ∼5600 proteins in which the proteins were fused at their C-termini to the IgG-binding domain of Protein A (Gelperin et al, 2005). In each case, the proteins were overexpressed in yeast and purified using the respective affinity tags. The proteome chips with the C-terminal protein fusions are ideal for screening type-I and the great majority of type-II and type-III membrane proteins that are glycosylated through the secretory pathway; these proteins have proper signal and translocation sequences and will be modified with N-linked glycans during or after translation. In contrast, proteome chips with the N-terminal protein fusions are ideal for the identification of proteins anchored to the membranes by a C-terminal signal sequence or transmembrane domain (e.g. cytochrome b5 and the SNARE proteins), as well as a portion of type-II proteins. Finally, both types of the yeast proteome chips are expected to detect O-linked glycoproteins, which do not have signal sequences for membrane transport. Fluorescently labelled lectins were incubated with the chips, and the slides were then washed to remove unbound lectins. The slides were scanned and images were processed using an algorithm adapted from ProCAT (Zhu et al, 2006). One problem with lectin probing is that the affinity of lectin–carbohydrate interactions is rather weak; the dissociation constants for lectin–glycan interactions in solution are approximately ∼300 μM for ConA–mannose and ∼2 mM for WGA–GlcNAc (Clegg et al, 1981; Mackiewicz and Mackiewicz 1986; Bains et al, 1992). In addition, some lectins often exhibit non-specific binding, either to the slide itself or to the proteins on the chip. Furthermore, lectins tend to be sticky proteins due to their biological function; they recognize glycans that differ little except in the conformations of small side chains. To circumvent the non-specific binding of ConA and WGA to the surfaces and 'sticky' protein spots while simultaneously maintaining strong signals, we developed a glycan competition assay that allowed for sensitive detection of specific glycans. For each lectin, two proteome slides were probed in parallel, one in the presence of the glycan and the other in its absence (Figure 1). For ConA and WGA, 10 mM α-methyl-mannoside and 50 mM chitin hydrolysate, respectively, were used as inhibitors. These concentrations are well above the dissociation constants for the lectin–glycan associations and the concentrations of lectins used in the probings, and thus should completely prevent glycan-specific binding to the surface. By comparing the lectin binding in the presence and absence of the glycan inhibitors, the glycan-dependent interactions can be identified. All experiments were performed in duplicate on both the N- and C-terminal fusion libraries. Figure 1.A schematic representation of glycan competition assays. Proteome arrays fabricated with proteins of various types are probed with fluorescently labelled lectins. (Left track) A proteome chip is incubated with lectins, followed by a washing step to remove free lectins and some weak, non-specific interactions; however, stronger non-specific interactions, presumably caused by protein–protein interactions, still remain, leaving behind glycan-independent binding signals. (Right track) In parallel, a proteome chip is incubated with lectins in the presence of excess amount of glycan competitors, which should block glycan-dependent interactions. The comparison of signal intensities between the two tracks reveals proteins that show glycan-dependent binding activities. Download figure Download PowerPoint Examples of the probings are shown in Figure 2. Although many proteins bound both in the presence and absence of the competing glycan inhibitors, a large number of proteins were identified that bound only in the absence of the competitors. All proteins recognized by the two lectins were identified and the glycoprotein candidates were determined as follows: set ORFx_Net Signal = ORFx_Signal (without sugar competitor)−ORFx_Signal (with sugar competitor), then the cut off is ORFx_Net Signal⩾3∗Stdev (ORFx_Net Signal: ORFn_Net Signal), n equal the number of ORFs on the proteome chip. It was interesting to note that more binding signals were observed on the C-terminal proteome chips, which might reflect the differences in the tag positions of the fusion proteins and/or surface chemistries of the two types of proteome chips. Figure 2.Glycoprotein identification using two types of proteome chips. (A) Lectin probing of the proteome chips. (Upper panel) C-terminal proteome chips probed with WGA–TAMRA (left) and ConA–Alexa647 (right). Many binding signals can be observed for both WGA and ConA in the absence of the sugar inhibitors; enlarged areas of the chips from both assays are illustrated in the middle. However, by comparing with the signals in the presence of sugar inhibitors (data not shown), 236 candidate glycoproteins were identified with ConA and 142 with WGA. (Lower panel) N-terminal proteome chips probed with WGA–TAMRA (left) and ConA–Alexa647 (right). Because of the N-terminal tags, less lectin binding signals were observed as expected. Using the same approach, ConA and WGA were found to identify 124 and 174 of positive proteins, respectively. (B) Examples of the sugar competition experiments. Representative glycoprotein candidates were shown. The inhibitors for WGA and ConA are chitin hydrolysate and α-methyl mannoside, respectively. Download figure Download PowerPoint Chip probing results and identification of glycoproteins Of the 5573 proteins on the proteome chips with C-terminal fusion proteins, 236 candidate glycoproteins were identified with ConA and 142 with WGA (Supplementary Table I). Of the 305 previously identified S. cerevisiae glycoproteins, 270 were present on the proteome chips, 105 were found by ConA, and 79 by WGA. Examples include DFG5 and YPS6. The glycoproteins that were not detected tended to be present at low levels on the arrays as evidenced by probing with antibodies to the tags. Nonetheless, these numbers represent very high ratio over the number expected by random selection of candidate proteins (9.56- and 12.05-fold, respectively) over that expected by chance. In addition, enrichment of proteins with predicted signal peptides is quite high (6.7- and 8.6-fold, respectively; Nielsen et al, 1997; Bendtsen et al, 2004). Thus, the candidate lists are highly enriched for glycoproteins and those with signal peptides. Because all N-linked glycoproteins are modified with both GlcNAc and mannose, it was expected that many of the WGA-identified candidates would also appear in the ConA probing. A total of 112 (79%) of WGA-binding protein also bound ConA. The failure to of ConA to detect all WGA-reactive proteins may arise from the stringent cut-offs used in the determination of the candidate lists, the accessibility of the different sugars to the lectins, or their suitable presentation on the protein array surface. Using the same protocol, ConA and WGA were used to probe the proteome chips printed with the N-terminal fusion proteins. Of the 5800 proteins, ConA and WGA identified 124 and 174 of positive proteins, respectively (Supplementary Table I). Of the 305 previously identified S. cerevisiae glycoproteins, 284 were on the N-terminal proteome chips and 15 were found by ConA. The enrichment ratio is about 2.5 over the number expected by random selection of candidate proteins. However, there is no enrichment for WGA; this is presumably because the GST tag located at the N-termini of the fusion proteins masks the N-terminal signal peptides in the type-I and the great majority of type-II and type-III membrane proteins. Gene ontology analysis of candidate glycoproteins The candidate glycoproteins were analyzed using gene ontology (GO) annotations to identify common and enriched properties of these proteins compared with the rest of the S. cerevisiae proteome. The combined ConA and WGA list of 266 unique candidate glycoproteins identified using the C-terminal fusion protein chips was examined for enrichment in cellular component, biological process, and molecular function compared with the GO annotations of the proteins in the Saccharomyces Genome Database (Fisk et al, 2006). For cellular component, the candidate list was substantially (P<10−8) enriched for chitin- and β-glucan-containing cell wall components, proteins in the ER and vacuole, as well as secreted proteins (Figure 3). There was also enrichment (P<0.01) for membrane proteins. Of the 113 known chitin- and β-glucan-containing cell wall proteins, 52 (46%) were found by the lectin screening, representing 12.6-fold enrichment. For the other enriched components, about 10–15% of the known proteins were identified, representing 2.5- to 4.5-fold enrichment. Figure 3.GO analysis of candidate glycoproteins. Chart of enriched GO annotations in the list of candidate glycoproteins when compared with the entire S. cerevisiae proteome. Each category listed is significantly enriched (with P-value <0.01 as shown), and both the number of candidates belonging to the annotation (blue) and the fold enrichment over the proteome (yellow) are shown. Download figure Download PowerPoint For biological process, the candidate list was substantially (P<10−12) enriched in cell wall organization and biogenesis, and more specifically (P<0.01) for chitin- and β-glucan-containing cell wall organization and biogenesis. The 36 candidates, involved in chitin- and β-glucan cell wall organization and biogenesis, represent 18% of the 199 proteins annotated to the category involved in the cell wall organization and biogenesis process, nearly fivefold enrichment. For molecular function, the candidate list was substantially (P<10−4) enriched in hydrolase activity (used in the hydrolysis of glycosyl bonds in O-glycosyl compounds), as well as cell wall structure and glucosidase activity (P<0.001), and (membrane) receptor activity (P<0.01). The large enrichment of cell wall components, processes, and function for C-terminal fusion proteins identified by lectin screening is expected, given the extensive glycosylation of these proteins. Enrichments were not observed for proteins identified from the N-terminal collection and might also be expected. These results validate that the lectin screen is indeed finding proteins, which are modified with glycans or interact with glycan chains. Validation of glycoprotein candidates by secondary gel-shift assay Candidate glycoproteins were further tested for glycosylation by determining if they exhibit a gel mobility shift after treatment with enzymes that remove glycan modifications (Figure 4 and Supplementary Figure 1). Purified candidate glycoproteins were subjected to digestion/treatment with two enzymes, which remove N-linked glycan chains, endoglycosidase Hf, and PNGase F. The digested proteins were compared with untreated samples by SDS–PAGE. Candidates showing a mobility difference between the two were scored as validated N-linked glycoproteins. The gel-shift assay had previously been shown to be quite robust, with 84% of known N-linked glycoproteins exhibiting shifts upon enzyme treatment and none of 19 negative controls showing shifts (Gelperin et al, 2005). Figure 4.Validation of glycosylation using gel-shift assays. Five representatives, for example, Ylr413wp, Ydr065wp, Ycl045cp, Lpe10p, and Yhr180wp, show the mobility of proteins digested with endoglycosidases (+) or without (−). The difference in mobility upon digestion indicates the cleavage of N-linked glycans from the protein, validating the candidate as a glycoprotein. Download figure Download PowerPoint Of 118 candidate C-terminal glycoproteins and 24 candidate N-terminal glycoproteins screened, 51 (36%) were validated as N-linked glycoproteins with the gel-shift assay, six of them were known glycosylated protein, which were used as positive controls. Some of the candidates tested did not give conclusive results because of difficulties in purification or the known limitations of the gel-shift assay, such as detection difficulties due to smearing of heterogeneous glycan chains. Nonetheless, these studies validated 45 new glycoproteins (listed in Table I) and extended the total number of validated N-linked glycoproteins in yeast to 276, and the total number of the validated glycoproteins in S. cerevisiae to 350. Table 1. Validated novel glycoproteins Systematic name Common name Cellular component YCR026C NPP1 Unknown YDR434W GPI17 Cytoplasm/endomembrane system/ endoplasmic reticulum/membrane YFR020W YFR020W Unknown YLR413W YLR413W Unknown YCL045C YCL045C Endoplasmic reticulum YCR045C YCR045C Unknown YDR065W YDR065W Mitochondrion YGR279C SCW4 Cell wall YHR039C MSC7 Endoplasmic reticulum YIL039W TED1 Endoplasmic reticulum YJL009W YJL009W Unknown YLR414C YLR414C Cellular bud/cytoplasm YMR215W GAS3 Membrane fraction/cell wall YNL080C EOS1 Membrane/endoplasmic reticulum/ endomembrane system/cytoplasm YNL283C WSC2 Site of polarized growth/membrane fraction/cellular bud/cytoplasm YOR387C YOR387C Other YBR298C-A YBR298C-A Unknown YCR089W FIG2 Cell wall YER158C YER158C Unknown YER183C FAU1 Mitochondrion YER185W PUG1 Membrane YGL020C GET1 Endoplasmic reticulum/cytoplasm YGR223C HSV2 Cytoplasm/vacuole YHR202W YHR202W Cytoplasm/other/vacuole YIL059C YIL059C Unknown YJL038C LOH1 Unknown YJL158C CIS3 Site of polarized growth/cell wall/ extracellular region/cellular bud/ plasma membrane/endoplasmic reticulum YLR337C VRP1 Cellular bud/site of polarized growth/ other/cell cortex/cytoskeleton/cytoplasm YMR238W DFG5 Membrane/plasma membrane YNR015W SMM1 Nucleus/cytoplasm YOL031C SIL1 Endoplasmic reticulum YOR099W KTR1 Golgi apparatus YOR214C YOR214C Cell wall YOR246C YOR246C Cytoplasm YOR292C YOR292C Cytoplasm/vacuole YPL060W LPE10 Cytoplasm/membrane/mitochondrial envelope/mitochondrion YPL277C YPL277C Membrane fraction YGL258W VEL1 Other YIR039C YPS6 Cell wall YDR088C SLU7 Nucleus YNL054W VAC7 Membrane/cytoplasm/vacuole YNL094W APP1 Cell cortex/cytoplasm/cytoskeleton YNL308C KRI1 Nucleolus YOR027W STI1 Cytoplasm YPL
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