The N-terminus of Vps74p is phosphorylated

To characterize the function of Vps74p, we first generated an antibody against Vps74p. Western blot analysis of wild-type yeast total cell lysates using our anti-Vps74p antibody showed two distinct bands that migrated closely (~39 to 41 kDa, the expected size for Vps74p); however, these bands were absent in lysates obtained from vps74Δ cells (Figure 1A). Vps74p has been shown to have at least three phosphorylation sites at serines 14, 19, and 23 [24]. To assess whether these two bands in the Western blot analysis represented the phosphorylated and non-phosphorylated forms of Vps74p, we used an anti-HA antibody to perform immunoprecipitation using lysates from yeast expressing HA-tagged Vps74P (WT) and calf intestine alkaline phosphatase (CIP)-treated immunoprecipitated HA-tagged Vps74p. As shown in Figure 1B, two bands were detected by Western blot analysis after immunoprecipitation. However, after CIP treatment, the lower mobility band disappeared. This suggests that the low mobility (upper) band might be a phosphorylated form of Vps74p.

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Figure 1. Vps74p is a phospho-protein.

(A) The anti-Vps74p antibody is specific, Western blot analyses of crude lysates of wild-type (WT) and vps74Δ mutant yeast probed with antibodies against Vps74p (right) and SDS-PAGE stained with Coomassie Blue (left) demonstrates the specificity of the Vps74p antibody. (B) Cell lysis and immunoprecipitation assays were performed as described in the Materials and methods section. Immunoprecipitated proteins were separated into two equal portions and incubated at 37°C in CIP buffer for 1 h in the absence or presence of CIP. Each sample was subjected to SDS-PAGE followed by Western blotting using anti-HA antibody. (C) Schematic representation of the structure of the 346-residue Vps74p polypeptide and various mutant constructs is illustrated. (D) and (E) Expression of wild-type and mutant proteins is dipicted. HA epitope-tagged wild-type (WT) Vps74p, Vps74p-dN66, Vps74p-dN90, Vps74p-dN122, Vps74p-dC83, and Vps74p-3pm were expressed from 2μ-based plasmids (pVT101U) under the control of the ADH promoter in BY4741 yeast strain and detected by western blotting using the indicated antibodies.

https://doi.org/10.1371/journal.pone.0074715.g001

To evaluate the significance of Vps74p S14, S19, and S23 phosphorylation in vivo, we constructed a HA-tagged triple point mutation of Vps74p (HA-Vps74p-3pm) that could not be phosphorylated at residues -14, -19, and -23 by replacing these serines with alanines. When this mutant was expressed in yeast using a high copy number plasmid, HA-Vps74p-3pm appeared to migrate as a single band; however, we observed two distinct bands representing the overexpressed wild-type HA-Vps74p. The mobility of the HA-Vps74p-3pm matched that of the higher mobility band of HA-Vps74p. We also evaluated CIP-treated immunoprecipitated HA-Vps74p-3pm and found that Vps74p-3pm produced only the lower band, regardless of whether the samples were subjected to CIP treatment (Figure 1B). These results confirmed that Vps74p is a phospho-protein, and this non-phosphorylatable Vps74p construct was used in further studies of Vps74p to characterize the function of Vps74p in yeast.

Structural analyses of Vps74p have indicated that Vps74p might form tetramers and that oligomerization is required for its Golgi localization [7]. Several regions within Vps74p contribute to oligomerization of the protein: α-helixes 1, 2, 9, and 10 and β-hairpins 3 and 4 [7]. To further characterize Vps74p functions in yeast, we constructed several truncated mutants of Vps74p. We truncated the N-terminal 66 (truncated before α-1), 90 (α1-deleted), or 122 (α1- and α2-deleted) amino acids or the C-terminal 83 amino acids (α9 through 12 deleted) (Figure 1C). Upon exogenous overexpression in yeast, overexpressed HA-Vps74p migrated as two distinct bands. As shown in Figure 1D, the three N-terminally truncated Vps74p mutants, HA-Vps74p-dN66, -dN90, and -dN122, migrated as one band when detected with anti-Vps74p and anti-HA antibodies, supporting the notion that Vps74p was phosphorylated at the N-terminus. HA-Vps74p-dC83 also migrated as one band, suggesting that the C-terminal deletion might affect the phosphorylation at the N-terminus of the protein.

The N-terminal domain (1–66) is dispensable for Vps74p localization to the Golgi

To examine whether phosphorylation is required for Vps74p localization to the Golgi and to identify the sequence elements that contribute this event, we examined the localization of non-phosphorylatable and truncated mutants of Vps74p. Wild-type and mutant forms of Vps74p were tagged with GFP at their N-termini and each of these constructs were co-transformed into a vps74Δ mutant yeast strains with either Arf1p-mRFP or Arl1p-mRFP, (Golgi markers that reside in the cis- or trans-Golgi, respectively) (Figure 2). Wild-type Vps74p partially co-localized with Arf1p and Arl1p (Figures 2 and S1), indicating that Vps74p localizes to both the cis- and trans-Golgi networks. Vps74p-dN66 and Vps74p-3pm, also exhibited partial co-localization with Arf1p and Arl1p, indicating that neither phosphorylation nor the N-terminal 66 amino acids are required for Golgi localization of Vps74p. However, GFP-tagged Vps74p-dC83, Vps74p-dN90, and Vps74p-dN122 signals were observed on some punctuate structures that did not co-localize with the Golgi markers. These findings clearly indicated that the N-terminal 66 amino acids are not required for the association of Vps74p with the Golgi.

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Figure 2. Vps74p is localized to the Golgi apparatus.

N-terminal GFP-fused-Vps74p, -Vps74p-dN66, -Vps74p-dN90, -Vps74p-dN122, -Vps74p-dC83, and -Vps74p-3pm from a 2μ vector (pVT101U) under an ADH promoter were transformed into vps74-deleted yeast cells containing Arf1p-mRFP. Mid-log phase cells were imaged live via microscopy. (B) The quantitative measurements of the Golgi co-localization are presented.

https://doi.org/10.1371/journal.pone.0074715.g002

The N-terminal 66 residues of Vps74p are required for its function on glycosyltransferase retention and Gas1p processing

One suggested functions of Vps74p is to maintain the proper localization of Golgi-resident mannosyltransferases. Mislocalization of Golgi mannosyltransferases would affect the modification and processing of many cellular proteins, similar to the effect of deleting the mannosyltransferases themselves. Deletion of KRE2 results in the hypoglycosylation of Gas1p, a plasma membrane β-1,3-glucanosyltransferase that is important for cell wall integrity [25,26]. Deletion of VPS74 or disruption of Vps74p tetramerization and Golgi localization in yeast also results in the under-modification of Gas1p [6]. Therefore, we examined whether the N-terminal 66 amino acids of Vps74p are essential for Gas1p processing. As shown in Figure 3A, cells lacking VPS74 displayed Gas1p processing defects. This activity was restored after overexpressing either full-length HA-Vps74p or the phosphorylation mutant HA-Vps74p-3pm. Overexpression of HA-Vps74p-dN66 or Vps74p-dC83 could not restore Gas1p processing in vps74Δ cells. These results indicate that both N- and C-terminal regions of Vps74p are required for Gas1p processing, however, the phosphorylation of Vps74p at the N terminus is not.

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Figure 3. The effects of Vps74p on cell wall integrity are not due to the mislocalization of glycosyltransferases.

(A) BY4741 wild-type, kre2Δ, and vps74Δ mutants were transformed with the empty vector pVT101U (Vec). BY4741 vps74Δ was transformed with HA-tagged Vps74, Vps74p-dN66, Vps74p-dC83, and Vps74p-3pm. Lysates prepared from these cells were subjected to Western blot analyses using an anti-Gas1p antibody. (B) and (C) The localization of Kre2p-GFP in vps74Δ mutant cells. HA-VPS74 and HA-VPS74-dN66, under the control of the ADH promoter in a 2μ plasmid, was transformed into vps74Δ mutant strains expressing Kre2p-GFP from its native locus. Transformants were grown in selection medium and the GFP signals were observed. Expression of Arf1p-mRFP was used as a Golgi marker. FM4-64 tracer dye was used for vacuole localization. (D) BY4741 was transformed with the empty vector pVT101U (Vec), and BY4741 vps74Δ yeast were transformed with pVT101U (Vec) and HA-tagged Vps74p, Vps74p-dN66, Vps74p-dN90, Vps74p-dN122, Vps74p-dC83, and Vps74p-3pm. These cells were cultured to mid-log phase. Subsequently, 10-fold serial dilutions were spotted onto Ura minus plates containing 2% glucose (left panel), YPD plates containing 50 µg/ml Congo red (middle panel), or YPD plates containing 50 µg/ml Congo red and 1.2 M sorbitol (right panel).

https://doi.org/10.1371/journal.pone.0074715.g003

Vps74p is involved in the specific retention of mannosyltransferases, such as Kre2p, at the cis- and medial-Golgi compartments by binding directly to their cytoplasmic tails. In vps74 mutant cells, Kre2p proteins are mislocalized to the vacuolar lumen [7]. We therefore examined whether the N-terminal 66 residues are required for the retention of Kre2p in the Golgi by assaying Kre2-mRFP in vps74Δ mutants expressing HA-Vps74p or HA-Vps74p-dN66. In vps74Δ cells overexpressing Vps74p, normal Golgi localization of Kre2p-mRFP was observed. However, in vps74Δ cells expressing Vps74p-dN66, Kre2p-mRFP did not localize to the Golgi (Figure 3B). These data indicated that the N-terminal 66-amino acid region of Vps74p is required for the retention of Kre2p in the Golgi. These results also suggested that Golgi localization alone is not sufficient for Vps74p to support the retention of Golgi mannosyltransferase and that different domains work in concert to execute the cellular function of Vps74p.

Deletion of specific genes involved in glycosylation events in yeast results in cell wall integrity defects and reduced viability in the presence of Calcofluor white (CW) or Congo red. Deletion of VPS74 also resulted in Calcofluor white sensitivity, most likely due to the mislocalization of glycosyltransferases and the subsequent under-modification of proteins involved in the cell wall integrity in vps74Δ mutant cells [6]. To determine if Vps74p mutants that are unable to rescue Kre2p mislocalization or Gas1p under-modification phenotypes are also unable to rescue the cell wall integrity defects in vps74Δ cells, we examined the viability of vps74Δ cells and vps74Δ cells expressing various Vps74p mutants in the presence of Congo red. As shown in Figure 3C, vps74Δ mutants were hypersensitive to Congo red and this hypersensitivity could be rescued by the addition of 1.2 M sorbitol to adjust the osmolarity. This finding confirmed that the vps74Δ mutant hypersensitivity to Congo red results from defects in cell wall integrity. Moreover, none of the Vps74p-truncated mutants that failed to localize to the Golgi (-dN90, -dN122, or -dC83) repressed the Congo red hypersensitivity of vps74Δ cells, although wild-type and the -3pm mutant proteins did localize to the Golgi. Unexpectedly, the hypersensitivity was also repressed when we overexpressed the dN66-truncated mutant of Vps74p, despite the inability of this mutant protein to facilitate proper Gas1p glycosylation. Thus, under-processed glycosylated proteins cannot account for the cell wall integrity defects in vps74Δ mutant cells (Figure 3D), suggesting that Vps74p might be responsible for other unidentified cellular processes involved in maintaining the cell wall integrity.

The N-terminal 66 residues of Vps74p are not required for cdc34-2-dependent apical growth

Genetic screens for genes involved in abnormal apical growth have identified VPS74 as one of the potential players in this process. Mutations in ubiquitin-dependent protein degradation pathways in yeast have been shown to induce abnormal apical growth, resulting in an elongated cell morphology [14,27,28]. Yeast cells harboring the temperature-sensitive CDC34 allele (cdc34-2), which contains a mutation in an E2 ubiquitin-conjugating enzyme gene, cannot degrade G1 cyclins or a G2 cyclin/cdk inhibitor at a restrictive temperature [11,13,29]. Under the restrictive temperature, these cells remain at a stage of constitutive apical growth, leading to the formation of multiple highly elongated buds [30]. Deletion of VPS74 in cdc34-2 cells has been shown to reduce the formation of elongated buds [4]. Therefore, we examined whether specific domains in Vps74p are required to alter the elongated bud formation of cdc34-2 cells at a restrictive temperature. Full-length VPS74, truncated constructs, and Vps74p-3pm were transformed into cdc34-2/vps74Δ mutants. As shown in Figure 4A, the buds in cdc34-2 cells exhibited an elongated cell morphology. Deletion of the VPS74 gene in cdc34-2 cells reversed this phenotype, and the buds retained their round shapes. Overexpressing wild-type Vps74p in cdc34-2/vps74Δ cells restored the elongated bud formation observed in the cdc34-2 mutant, indicating that Vps74p contributes to the formation of enlarged buds in cdc34-2 cells. Upon overexpression of the different truncation and phosphorylation mutants in cdc34-2/vps74Δ cells, only cells expressing Vps74p-3pm or Vps74p-dN66 demonstrated elongated bud formation at a restrictive temperature. Cells expressing Vps74p-dN90, -dN122, or -dC83 did not exhibit elongated bud formation. These results indicate that Golgi-associated Vps74p-dN66 might retain certain aspects of Vps74p function that are required for apical growth. Our findings also indicate that phosphorylation of Vps74p at the three N-terminal serine sites is not required for this function.

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Figure 4. The N-terminal 66 residues of Vps74p are not required for cdc34-2-dependent apical growth.

(A) Morphology transformants carrying the plasmid pVT101U (Vec) in cdc34-2 mutant were detected. Transformed cells were grown in a synthetic selection medium at 25°C for 2 h and then shifted to 37°C for 6 h. Next, the ability to complement the elongating morphology was investigated using microscopy. Cells of cdc34-2 with VPS74 deleted were transformed with the indicated plasmids and then imaged as previously described. (B) Yeast strains cdc34-2, pre1-1/pre4-1, cdc53-1, cdc4-, JY25, cdc34-2/vps74Δ, pre1-1/pre4-1/vps74Δ, cdc53-1/vps74Δ, cdc4-/vps74Δ, and JY25/vps74Δ were grown to mid-log phase and these cultures were then transferred from room temperature to 37°C for 6 h and fixed at 37°C. The morphologies of these cells were visualized using microscopy.

https://doi.org/10.1371/journal.pone.0074715.g004

Vps74p is specifically involved in cdc34-dependent polarized apical growth

Yeast with mutations in several other proteins involved in cell cycle progression also form elongated buds. For example, mutations in ubiquitin-dependent protein degradation pathways (cdc34, cdc53, and pre1-1/pre4-1) in yeast induce abnormal apical growth, resulting in elongated cell morphologies [28]. Mutations in CDC4, an F-box protein required for G1/S and G2/M transitions, lead to the formation of elongated buds [14]. In addition, the great majority of clbl,2,3,4 mutant cells (JY25 yeast strains) arrest in S phase and show a cellular elongation defect at non-permissive temperatures [8,31]. To evaluate whether VPS74 also interacts with these genes during elongated bud formation, we disrupted the VPS74 gene in pre1-1/pre4-1, cdc53-1, cdc4- and JY25 (clb1::URA3, clb2^ts, clb3::TRP1, clb4::HIS3) yeast strains. As shown in Figure 4B, when compared to the control parental strains, pre1-1/pre4-1/vps74Δ and cdc53-1/vps74Δ mutants exhibited a decreased degree of elongated bud formation. In contrast, the majority of the cdc4/vps74Δ and JY25/vps74Δ cells continued to form elongated buds. These results suggest that Vps74p is specifically involved in cdc34-dependent polarized apical growth.

We next examined whether Vps74p contributes to elongated bud formation via its ability to retain glycosyltransferases at the Golgi, which assures proper glycosylation of cell wall proteins. We deleted two glycosyltransferase genes, KRE2 and MNN1, and the cell wall protein GAS1 in cdc34-2, cdc4, or JY25 cells and evaluated if the formation of elongated buds was altered. At non-permissive temperatures, kre2Δ and mnn1Δ mutations did not alter elongated bud formation in any of these cells (Figure S2). Because Gas1p is under-glycosylated in vps74Δ cells (Figure 3A), we speculated that the gas1Δ mutation might specifically alter elongated bud formation in cdc34-2 cells, similar to vps74Δ. However, GAS1 deletion reversed the elongated bud formation phenotype in all three mutant yeasts strains examined, indicating that Gas1p-mediated cell wall biosynthesis broadly affects apical growth elongation. Thus, these data indicate that the mislocalization of glycosyltransferases or under-modification of cell wall proteins, such as Gas1p, may not be the primary causes of vps74Δ mutation-dependent morphological changes, indicating that other putative functions of Vps74p might be involved in these processes.

The PtdIns(4)P phosphatase Sac1p is involved in cdc34-2-dependent abnormal apical growth

Vps74p can modulate Golgi PtdIns(4)P homeostasis via interaction with the PtdIns(4)P phosphatase Sac1 [16]. Sac1p has been implicated in the coordination of cytoskeletal and secretory activities, and SAC1 deletion leads to defects in the cell wall integrity pathway [32]. To evaluate whether this aspect of Vps74p function contributes to elongated bud formation, we tested whether deletion of SAC1 affects elongated bud formation in cdc34-2 cells. At non-permissive temperatures, cdc34-2/sac1Δ, pre1-1/pre4-1/sac1Δ, and cdc53-1/sac1Δ cells did not form elongated buds, similar to cdc34-2/vps74Δ cells (Figure 5). In addition, SAC1 deletion did not affect the apical growth of cdc4- and JY25 mutants or Gas1p processing (data not shown). These results suggest that Sac1p is specifically involved in Cdc34p-regulated apical growth but does not retain glycosyltransferases at the Golgi.

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Figure 5. Sac1p is involved in cdc34-2-dependent apical growth.

Yeast strains cdc34-2, pre1-1 pre4-1, cdc53-1, cdc4-, JY25, cdc34-2/vps74Δ, pre1-1 pre4-1/vps74Δ, cdc53-1/vps74Δ, cdc4-/vps74Δ, JY25/vps74Δ, cdc34-2/sac1Δ, pre1-1 pre4-1/sac1Δ, cdc53-1/sac1Δ, cdc4-/sac1Δ, and JY25/sac1Δ were grown to mid-log phase and then transferred to 37^oC for 6 h. The morphologies of these cells were visualized using microscopy.

https://doi.org/10.1371/journal.pone.0074715.g005

Arf1p participates in the abnormal apical growth of the cdc34-2 mutant

Vps74p functions as an adaptor protein for COPI to recruit Golgi-resident glycosyltransferases into COPI-coated vesicles [6]. However, Vps74p-dN66, which cannot mediate Golgi glycosyltransferase recruitment into COPI-coated vesicles [33], reversed the phenotype of cdc34-2/vps74Δ cells (Figure 4A). We therefore examined whether the disruption of COPI vesicle formation affects apical growth in cdc34-2 cells. The formation of COPI-coated vesicles is regulated by the small GTPase Arf1p. Arf1p is a member of ARF family of proteins that regulate coated vesicle formation. We examined whether deletion of ARF1 in cdc34-2 cells affects elongated bud formation. At a restrictive temperature, cdc34-2/arf1Δ cells did not form elongated buds, similar to cdc34-2/vps74Δ cells (Figure 6A). Two ARF family members, Arl1p and Arl3p, are known to regulate the specific vesicular transport pathway at the trans-Golgi. Therefore, we examined whether these two molecules also participated in the formation of elongated buds in cdc34-2 cells. Neither Arl1p nor Arl3p were required for the elongated bud phenotype in cdc34-2 cells (Figure 6A), indicating that Arf1p, but not Arl1p or Arl3p, is involved in cdc34-2-dependent elongated bud formation. To investigate whether Arf1p regulates cdc34-2-dependent apical growth, we also examined the morphology of cdc4- and JY25 mutant cells containing an arf1Δ or arl1Δ mutation at non-permissive temperatures (Figure 6B). Neither the arf1Δ nor arl1Δ mutation affected apical growth in cdc4- and JY25 mutants. VPS74 has been shown to genetically interact with several proteins involved in retrograde transport pathways, including PEP8 and YPT6. Pep8p is a vacuolar sorting protein that is essential for endosome-to-Golgi retrograde protein transport [34] and was identified as a Vps74p-binding partner in a comprehensive two-hybrid analysis [35]. Ypt6p is a Ras-like GTP-binding protein involved in the secretory pathway and is required for fusion of endosome-derived vesicles with the late Golgi [36,37] and has shown synthetic lethality with VPS74 [3]. We therefore examined whether these molecules also contribute to abnormal bud formation in cdc34-2 cells. Upon disruption of PEP8 or YPT6 in cdc34-2 mutants, the cells formed elongated buds at a restrictive temperature, indicating that the interaction of these genes with VPS74 might not contribute to this phenotype (Figure 6A). Taken together, these results show that although the Vps74p-COPI interaction is not required for the role of Vps74p in mediating elongated bud formation, COPI vesicle formation or other Arf1p-dependent pathways are required for elongated bud formation in cdc34-2 cells.

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Figure 6. Arf1p facilitates elongated bud formation in cdc34-2 mutant.

(A) cdc34-2, cdc34-2/vps74Δ, cdc34-2/arf1Δ, cdc34-2/arl1Δ, cdc34-2/arl3Δ, cdc34-2/pep8Δ, and cdc34-2/ypt6Δ mutants (B) cdc4-/arf1Δ, cdc34-2/arl1Δ, JY25/arf1Δ, and JY25/arl1Δ mutants were grown in YPD at 25°C to an OD600 nm of 0.2, shifted to 37°C for 6 h, and imaged by microscopy.

https://doi.org/10.1371/journal.pone.0074715.g006

Double deletion of VPS74 and ARF1 results in a synthetic cell wall integrity defect

VPS74 and ARF1 both modulate COPI-coated vesicle formation and similar phenotypes are observed upon deletion of either gene in cdc34-2 cells. We therefore examined whether ARF1 and VPS74 functioned in concert or in parallel to facilitate these cellular processes. Arf1p is known to contribute to the maintenance of cell wall integrity. We first examined whether vps74Δ and arf1Δ double mutants were hypersensitive to Congo red. As shown in Figure 7A, similar to kre2Δ and gas1Δ mutants, both vps74Δ and arf1Δ mutants were sensitive to Congo red, and double deletion of VPS74 and ARF1 displayed a synthetic hypersensitivity to Congo red. This hypersensitivity could be partially rescued by expressing either Vps74p or Arf1p from low-copy CEN vectors, but not by osmolarity adjustment (addition of 1.2 M sorbitol), suggesting that the vps74arf1Δ double deletion mutant has a severe defect in cell wall integrity (Figure 7B). This result suggests that the function of Vps74p and Arf1p are related, in part, to different transport processes involved in cell wall integrity. Although deletion of another Golgi small GTPase, Arl1p, also resulted in Congo red sensitivity, the arl1vps74Δ double mutant did not exhibit a synthetic hypersensitivity to Congo red. We also examined whether Arf1p was required for Gas1p modification. Western blot analysis of Gas1p modification in arf1Δ cells revealed that, unlike vps74Δ cells, the Gas1p glycosylation was not affected in arf1Δ cells (Figure 7C). Taken together, these results indicated that Vps74p and Arf1p might function in parallel pathways contributing to apical growth and the maintenance of cell wall integrity.

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Figure 7. Deletion of VPS74 in combination with ARF1 results in a synergistic defect in cell wall integrity.

(A) and (B) Indicated cells were cultured to mid-log phase, then 10-fold serial dilutions were spotted onto Ura minus plates containing 2% glucose (left panel), YPD plates containing 50 µg/ml Congo red (middle panel), or YPD plates containing 50 µg/ml Congo red and 1.2 M sorbitol (right panel). (C) Lysates were prepared from the indicated cells and proteins were precipitated with TCA to prepare an immunoblot that was probed with anti-Gas1p polyclonal antibodies.

https://doi.org/10.1371/journal.pone.0074715.g007

Phosphorylation of Vps74p contributes to rapamycin hypersensitivity

Recent studies have shown that overexpression of mammalian Vps74p and GOLPH3 results in cell transformation, tumor growth in vivo, and rapamycin hypersensitivity [20]. We examined the rapamycin sensitivity of yeast cells overexpressing Vps74p and found a similar effect: overexpression of full-length Vps74p resulted in rapamycin hypersensitivity. We next expressed various Vps74p truncated and non-phosphorylatable mutants to examine which region of Vps74p might be responsible for this phenotype. We found that none of the yeast cells expressing these mutants displayed rapamycin hypersensitivity, including Vps74p-3pm (Figure S3). These data suggested that the Golgi localization and phosphorylation of Vps74p contribute to rapamycin hypersensitivity. The effect of GOLPH3 on mTOR signaling has also been suggested to be mediated through the interaction of GOLPH3 with VPS35 to regulate retrograde transport and receptor recycling of key molecules [20]. We therefore examined the genetic interaction between VPS74 and VPS35 in yeast. High throughput screening data have shown that VPS74 and VPS35 both demonstrate synthetic lethality with YPT6 deletion in yeast [3]. We overexpressed Vps74p in vps35Δ or ypt6Δ cells and found that overexpression of Vps74p did not enhance rapamycin hypersensitivity in either mutant cells (Figure S4). These results suggested that the rapamycin hypersensitivity observed upon Vps74p overexpression is dependent on Ypt6p- or Vps35p-modulated processes. Therefore, yeast Vps74p may play a similar role to that of mammalian VPS74-family proteins in inducing rapamycin hypersensitivity. Both the function of Vps74p in retrograde transport and phosphorylation at the N-terminal domain of the protein contribute to the rapamycin hypersensitivity.

Strains, media, and microbiological techniques

Table S1 lists the yeast strains used in this study. Yeast culture media were prepared as previously described [15]. YPD contained 1% Bacto-yeast extract, 2% Bacto-peptone, and 2% glucose. SD contained 0.2% Difco yeast nitrogen base (without amino acid), 0.5% ammonium persulfate, and 2% glucose. Nutrients essential for the auxotrophic strains were supplied at the specified concentrations. Yeast strains were transformed using the lithium acetate method [40]. Plasmids listed in Table S2 were constructed according to standard protocols. Gene disruption was performed as previously described [41].

Preparation of yeast cell extracts and Western blot analyses

Whole yeast extracts were prepared by agitating yeast cells suspended in TE buffer (10 mM Tris, pH 7.4, 1 mM EDTA) with glass beads for 1 min followed by incubation on ice for 1 min, and this was repeated 5 times. After a brief centrifugation to clarify the lysate, the protein was quantified using the Coomassie blue assay (Pierce). Proteins separated by SDS-PAGE were transferred onto PVDF membranes (Millipore), which were incubated (60 min, room temperature) with antibodies in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 and 5% dried skim milk. Bound antibodies were detected using the ECL system (Amersham Pharmacia Biotech).

Expression and purification of recombinant proteins and polyclonal antibody production

To create the His-tagged Vps47p, a DNA fragment containing the VPS74 coding region was generated after amplifying yeast genomic DNA using sequence-specific primers. The PCR product was purified and ligated to the expression vector pET30a (Novagen), yielding pET30a/Vps74p. The His-tagged fusion protein was synthesized in BL21(DE3) E. coli and purified on a Ni²⁺-NTA resin (Qiagen, Chatsworth, CA) as previously described [42]. Denatured, purified recombinant Vps74p isolated from an SDS-PAGE gel was used as an antigen to raise polyclonal antibodies in rabbits, as described previously [42]. The polyclonal antibodies were diluted 1:5000 for Western blotting analyses.

Immunoprecipitation and phosphatase treatment

Yeast cells were harvested by centrifugation and washed once with ddH₂O, and broken by vortexing with glass beads. The total cell protein extracts were obtained by precipitation with 4% (vol/vol) trichloroacetic acid (TCA), washed twice by cold ddH₂O, solubilized in buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1% SDS, and then denatured in 95°C for 5 min. The soluble fraction was then collect by centrifugation at 4°C. The volume of soluble fraction was brought to 1 ml with buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton-X 100, 1 mM DTT, and protease inhibitor and incubate with anti-HA conjugated agarose beads for 2 h at 4°C on rotator. The anti-HA beads-immune complex was then pelleted by centrifugation, washed three times with dilution buffer, separated into two portions to incubate in CIP buffer in the presence or absence of calf intestinal phosphatase (CIP) for 1 h at 37°C on a rotator. The beads were then washed twice in dilution buffer, suspended in SDS-PAGE sample buffer, and boiled for 10 min prior to separation by SDS-PAGE and Western blotting analyses.

Microscopy

The morphologies of living cells were observed after overnight culture to the mid-log phase. Yeast cells were grown at 25°C for 2 h and then at 37°C for 6 h. The ability to complement the elongating morphology was investigated using a Zeiss Axioskop microscope equipped with a Cool Snap FX camera.