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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 a phospho-protein.(A) The anti-Vps74p antibody is specific, Western blot analyses of crude lysates of wild-type (WT) and vps74Δ mutant yeast probed with antibodies against Vps74p (right) and SDS-PAGE stained with Coomassie Blue (left) demonstrates the specificity of the Vps74p antibody. (B) Cell lysis and immunoprecipitation assays were performed as described in the Materials and methods section. Immunoprecipitated proteins were separated into two equal portions and incubated at 37°C in CIP buffer for 1 h in the absence or presence of CIP. Each sample was subjected to SDS-PAGE followed by Western blotting using anti-HA antibody. (C) Schematic representation of the structure of the 346-residue Vps74p polypeptide and various mutant constructs is illustrated. (D) and (E) Expression of wild-type and mutant proteins is dipicted. HA epitope-tagged wild-type (WT) Vps74p, Vps74p-dN66, Vps74p-dN90, Vps74p-dN122, Vps74p-dC83, and Vps74p-3pm were expressed from 2μ-based plasmids (pVT101U) under the control of the ADH promoter in BY4741 yeast strain and detected by western blotting using the indicated antibodies.
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pone-0074715-g001: Vps74p is a phospho-protein.(A) The anti-Vps74p antibody is specific, Western blot analyses of crude lysates of wild-type (WT) and vps74Δ mutant yeast probed with antibodies against Vps74p (right) and SDS-PAGE stained with Coomassie Blue (left) demonstrates the specificity of the Vps74p antibody. (B) Cell lysis and immunoprecipitation assays were performed as described in the Materials and methods section. Immunoprecipitated proteins were separated into two equal portions and incubated at 37°C in CIP buffer for 1 h in the absence or presence of CIP. Each sample was subjected to SDS-PAGE followed by Western blotting using anti-HA antibody. (C) Schematic representation of the structure of the 346-residue Vps74p polypeptide and various mutant constructs is illustrated. (D) and (E) Expression of wild-type and mutant proteins is dipicted. HA epitope-tagged wild-type (WT) Vps74p, Vps74p-dN66, Vps74p-dN90, Vps74p-dN122, Vps74p-dC83, and Vps74p-3pm were expressed from 2μ-based plasmids (pVT101U) under the control of the ADH promoter in BY4741 yeast strain and detected by western blotting using the indicated antibodies.

Mentions: To characterize the function of Vps74p, we first generated an antibody against Vps74p. Western blot analysis of wild-type yeast total cell lysates using our anti-Vps74p antibody showed two distinct bands that migrated closely (~39 to 41 kDa, the expected size for Vps74p); however, these bands were absent in lysates obtained from vps74Δ cells (Figure 1A). Vps74p has been shown to have at least three phosphorylation sites at serines 14, 19, and 23 [24]. To assess whether these two bands in the Western blot analysis represented the phosphorylated and non-phosphorylated forms of Vps74p, we used an anti-HA antibody to perform immunoprecipitation using lysates from yeast expressing HA-tagged Vps74P (WT) and calf intestine alkaline phosphatase (CIP)-treated immunoprecipitated HA-tagged Vps74p. As shown in Figure 1B, two bands were detected by Western blot analysis after immunoprecipitation. However, after CIP treatment, the lower mobility band disappeared. This suggests that the low mobility (upper) band might be a phosphorylated form of Vps74p.


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 a phospho-protein.(A) The anti-Vps74p antibody is specific, Western blot analyses of crude lysates of wild-type (WT) and vps74Δ mutant yeast probed with antibodies against Vps74p (right) and SDS-PAGE stained with Coomassie Blue (left) demonstrates the specificity of the Vps74p antibody. (B) Cell lysis and immunoprecipitation assays were performed as described in the Materials and methods section. Immunoprecipitated proteins were separated into two equal portions and incubated at 37°C in CIP buffer for 1 h in the absence or presence of CIP. Each sample was subjected to SDS-PAGE followed by Western blotting using anti-HA antibody. (C) Schematic representation of the structure of the 346-residue Vps74p polypeptide and various mutant constructs is illustrated. (D) and (E) Expression of wild-type and mutant proteins is dipicted. HA epitope-tagged wild-type (WT) Vps74p, Vps74p-dN66, Vps74p-dN90, Vps74p-dN122, Vps74p-dC83, and Vps74p-3pm were expressed from 2μ-based plasmids (pVT101U) under the control of the ADH promoter in BY4741 yeast strain and detected by western blotting using the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760917&req=5

pone-0074715-g001: Vps74p is a phospho-protein.(A) The anti-Vps74p antibody is specific, Western blot analyses of crude lysates of wild-type (WT) and vps74Δ mutant yeast probed with antibodies against Vps74p (right) and SDS-PAGE stained with Coomassie Blue (left) demonstrates the specificity of the Vps74p antibody. (B) Cell lysis and immunoprecipitation assays were performed as described in the Materials and methods section. Immunoprecipitated proteins were separated into two equal portions and incubated at 37°C in CIP buffer for 1 h in the absence or presence of CIP. Each sample was subjected to SDS-PAGE followed by Western blotting using anti-HA antibody. (C) Schematic representation of the structure of the 346-residue Vps74p polypeptide and various mutant constructs is illustrated. (D) and (E) Expression of wild-type and mutant proteins is dipicted. HA epitope-tagged wild-type (WT) Vps74p, Vps74p-dN66, Vps74p-dN90, Vps74p-dN122, Vps74p-dC83, and Vps74p-3pm were expressed from 2μ-based plasmids (pVT101U) under the control of the ADH promoter in BY4741 yeast strain and detected by western blotting using the indicated antibodies.
Mentions: To characterize the function of Vps74p, we first generated an antibody against Vps74p. Western blot analysis of wild-type yeast total cell lysates using our anti-Vps74p antibody showed two distinct bands that migrated closely (~39 to 41 kDa, the expected size for Vps74p); however, these bands were absent in lysates obtained from vps74Δ cells (Figure 1A). Vps74p has been shown to have at least three phosphorylation sites at serines 14, 19, and 23 [24]. To assess whether these two bands in the Western blot analysis represented the phosphorylated and non-phosphorylated forms of Vps74p, we used an anti-HA antibody to perform immunoprecipitation using lysates from yeast expressing HA-tagged Vps74P (WT) and calf intestine alkaline phosphatase (CIP)-treated immunoprecipitated HA-tagged Vps74p. As shown in Figure 1B, two bands were detected by Western blot analysis after immunoprecipitation. However, after CIP treatment, the lower mobility band disappeared. This suggests that the low mobility (upper) band might be a phosphorylated form of Vps74p.

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