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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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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.
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pone-0074715-g004: 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.

Mentions: 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.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760917&req=5

pone-0074715-g004: 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.
Mentions: 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.

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