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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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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.
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pone-0074715-g002: 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.

Mentions: 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.


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)

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.
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Related In: Results  -  Collection

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

pone-0074715-g002: 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.
Mentions: 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.

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