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The N-terminus of Vps74p is essential for the retention of glycosyltransferases in the Golgi but not for the modulation of apical polarized growth in Saccharomyces cerevisiae.

Hsu JW, Chang LC, Jang LT, Huang CF, Lee FJ - PLoS ONE (2013)

Bottom Line: We found that the N-terminal 66 amino acids of Vps74p are dispensable for its Golgi localization and modulation of cell wall integrity but are required for glycosyltransferase retention and glycoprotein processing.Deletion of Sac1p and Arf1p also specifically reduced the abnormal elongated bud phenotype in cdc34-2 cells.Thus, we propose that Vps74p may use different domains to interact with specific effectors thereby differentially modulating a variety of cellular functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Vps74p is a member of the PtdIns(4)P-binding protein family. Vps74p interacts with Golgi-resident glycosyltransferases and the coat protein COPI complex to modulate Golgi retention of glycosyltransferases and with the PtdIns(4)P phosphatase Sac1p to modulate PtdIns(4)P homeostasis at the Golgi. Genetic analysis has shown that Vps74p is required for the formation of abnormal elongated buds in cdc34-2 cells. The C-terminal region of Vps74p is required for Vps74p multimerization, Golgi localization, and glycosyltransferase interactions; however, the functional significance of the N-terminal region and three putative phosphorylation sites of Vps74p have not been well characterized. In this study, we demonstrate that Vps74p executes multiple cellular functions using different domains. We found that the N-terminal 66 amino acids of Vps74p are dispensable for its Golgi localization and modulation of cell wall integrity but are required for glycosyltransferase retention and glycoprotein processing. Deletion of the N-terminal 90 amino acids, but not the 66 amino acids, of Vps74p impaired its ability to restore the elongated bud phenotype in cdc34-2/vps74Δ cells. Deletion of Sac1p and Arf1p also specifically reduced the abnormal elongated bud phenotype in cdc34-2 cells. Furthermore, we found that three N-terminal phosphorylation sites contribute to rapamycin hypersensitivity, although these phosphorylation residues are not involved in Vps74p localization, ability to modulate glycosyltransferase retention, or elongated bud formation in cdc34-2 cells. Thus, we propose that Vps74p may use different domains to interact with specific effectors thereby differentially modulating a variety of cellular functions.

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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.
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pone-0074715-g006: 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.

Mentions: 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.


The N-terminus of Vps74p is essential for the retention of glycosyltransferases in the Golgi but not for the modulation of apical polarized growth in Saccharomyces cerevisiae.

Hsu JW, Chang LC, Jang LT, Huang CF, Lee FJ - PLoS ONE (2013)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760917&req=5

pone-0074715-g006: 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.
Mentions: 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.

Bottom Line: We found that the N-terminal 66 amino acids of Vps74p are dispensable for its Golgi localization and modulation of cell wall integrity but are required for glycosyltransferase retention and glycoprotein processing.Deletion of Sac1p and Arf1p also specifically reduced the abnormal elongated bud phenotype in cdc34-2 cells.Thus, we propose that Vps74p may use different domains to interact with specific effectors thereby differentially modulating a variety of cellular functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Vps74p is a member of the PtdIns(4)P-binding protein family. Vps74p interacts with Golgi-resident glycosyltransferases and the coat protein COPI complex to modulate Golgi retention of glycosyltransferases and with the PtdIns(4)P phosphatase Sac1p to modulate PtdIns(4)P homeostasis at the Golgi. Genetic analysis has shown that Vps74p is required for the formation of abnormal elongated buds in cdc34-2 cells. The C-terminal region of Vps74p is required for Vps74p multimerization, Golgi localization, and glycosyltransferase interactions; however, the functional significance of the N-terminal region and three putative phosphorylation sites of Vps74p have not been well characterized. In this study, we demonstrate that Vps74p executes multiple cellular functions using different domains. We found that the N-terminal 66 amino acids of Vps74p are dispensable for its Golgi localization and modulation of cell wall integrity but are required for glycosyltransferase retention and glycoprotein processing. Deletion of the N-terminal 90 amino acids, but not the 66 amino acids, of Vps74p impaired its ability to restore the elongated bud phenotype in cdc34-2/vps74Δ cells. Deletion of Sac1p and Arf1p also specifically reduced the abnormal elongated bud phenotype in cdc34-2 cells. Furthermore, we found that three N-terminal phosphorylation sites contribute to rapamycin hypersensitivity, although these phosphorylation residues are not involved in Vps74p localization, ability to modulate glycosyltransferase retention, or elongated bud formation in cdc34-2 cells. Thus, we propose that Vps74p may use different domains to interact with specific effectors thereby differentially modulating a variety of cellular functions.

Show MeSH
Related in: MedlinePlus