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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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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.
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pone-0074715-g007: 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.

Mentions: 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.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0074715-g007: 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.
Mentions: 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.

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