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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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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 37oC for 6 h. The morphologies of these cells were visualized using microscopy.
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pone-0074715-g005: 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 37oC for 6 h. The morphologies of these cells were visualized using microscopy.

Mentions: 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.


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)

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 37oC for 6 h. The morphologies of these cells were visualized using microscopy.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760917&req=5

pone-0074715-g005: 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 37oC for 6 h. The morphologies of these cells were visualized using microscopy.
Mentions: 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.

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