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Vid22p, a novel plasma membrane protein, is required for the fructose-1,6-bisphosphatase degradation pathway

C. Randell Brown*, Jameson A. McCann*, Graham Guo-Chiuan Hung, Christopher P. Elco and Hui-Ling Chiang{ddagger}

Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, 500 University Drive, Hershey, PA 17033, USA



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  		Fig. 1. The VID22 gene is necessary for FBPase degradation. (A) Wild-type cells, the vid22-1 transposon mutants and vid22-1 mutants transformed with the VID22 gene were grown in synthetic medium containing low glucose for 2 days to induce FBPase. FBPase degradation was examined after cells were shifted to medium containing 2% glucose for 0, 60 and 120 minutes. (B) FBPase production was induced in wild-type cells, {Delta}vid22 mutants and {Delta}vid22 mutants transformed with the VID22 gene. These cells were shifted to glucose for t=0, 30, 60, 120 and 180 minutes and the degradation of FBPase was examined.

 


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  		Fig. 2. FBPase accumulates in the cytosol in the {Delta}vid22 mutant. (A) The {Delta}vid22 mutant was glucose starved and then shifted to glucose for t=0 or t=60 minutes prior to harvest. Total lysates were subjected to proteinase K (PK) digestion in the absence or presence of 2% Triton X-100 (TX). (B) Both {Delta}vid22 and {Delta}vid24 strains were glucose starved and then shifted to glucose-rich media for 60 minutes. Cells were homogenized and subjected to differential centrifugation. The percentage of FBPase found in each fraction is indicated. (C) Wild-type cells and the {Delta}vid22 mutant strains were shifted to glucose for 0, 20 and 60 minutes and harvested. Total lysates were immunoblotted with anti-Vid24p antibodies. (D) Wild-type and {Delta}vid22 mutant strains were shifted to glucose for 30 minutes and harvested. Differential centrifugation was performed. Proteins from the total (T), high-speed supernatant (S) and high-speed pellet (P) fractions were immunoblotted with anti-Vid24p antibodies.

 


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  		Fig. 3. VID22 is not required for other vacuolar trafficking pathways. Wild-type, {Delta}pep4, {Delta}agp1 and {Delta}vid22 strains were grown to log phase and examined for the processing of API and CPY. (A) Processing of API in wild-type (lane 1), {Delta}pep4 (lane 2), {Delta}agp1 (lane 3) and {Delta}vid22 (lane 4) strains. (B) Processing of CPY in wild-type (lane 1), {Delta}pep4 (lane 2), {Delta}agp1 (lane 3) and {Delta}vid22 (lane 4) strains. (C) The degradation of peroxisomes in response to glucose was examined using thiolase as a marker protein. Peroxisomes were degraded with similar kinetics in both the wild-type and {Delta}vid22 strains. (D) Wild-type, {Delta}agp1 and {Delta}vid22 strains were incubated for 6 hours in SD(-N) containing 1 mm PMSF to induce the autophagy pathway. The accumulation of autophagic bodies was observed by light microscopy.

 


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  		Fig. 4. Vid22p is a glycoslyated integral membrane protein. (A) The predicted amino acid sequence of Vid22p based upon the DNA sequence of YLR373C. Vid22p is a 901 amino acid protein with a predicted molecular weight of 102 kDa and pI of 5.15. Vid22p contains 12 potential N-linked glycosylation sites (underlined regions) and one potential transmembrane domain (bold underlined region). (B) The VID22 gene was fused with a V5 coding sequence and integrated into a wild-type yeast strain. Vid22p-V5 was detected using SDS-PAGE and western blot analysis with antibodies directed against V5. Vid22p migrated as a doublet representative of glycosylated and unglycosylated forms of Vid22p (lane 1). Lysates were treated with or without endoH to determine whether Vid22p was glycosylated. (C) Cells were lysed in Con A binding buffer and incubated in the presence of Con A beads. The total, bound and unbound materials were examined for the presence of Vid22p, CPY or FBPase via western blot analysis. (D) Cells expressing Vid22p-V5 were grown in low glucose media for 2 days and then shifted to glucose rich media for 0, 60 and 120 minutes. Cells were lysed and examined for the levels of Vid22p, FBPase, Vid24p and Pma1p via western blot analysis. (E) Wild-type cells expressing Vid22p-V5 were labeled in ethanol and chased in the presence of 2% ethanol or 2% glucose. Cells were harvested at 0, 1 and 2 hours and total lysates were immunoprecipitated with anti-V5 antibodies. Radiolabeled Vid22p-V5 was visualized using a phosphorimager. (F) Vid22p-V5 cells were shifted to glucose for 30 minutes. Cells were homogenized and lysates were resuspended in either TE alone, TE containing Na2CO3 (ph 11.5) or TE containing 2% TX-100. The distribution of Vid22p and Pma1p in the pellet and the supernatant fractions was examined by western blotting with anti-V5 or anti-Pma1p antibodies.

 


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  		Fig. 5. Vid22p sediments in a low-speed fraction. The Vid22p-V5 strain was glucose starved and then shifted to glucose containing media for 30 minutes. Differential centrifugation of the yeast cellular lysate was used to determine the localization of Vid22p (a). The majority of Vid22p was found to localize within the P1 (low-speed) fraction. A small amount of Vid22p was detected in the P13 fraction. The cytosolic marker enolase was found in the S200 fraction (b). The vacuole marker DPAP B and endosomal marker Pep12p were found primarily in the P13 fraction (c-d). The plasma membrane ATPase Pma1p was localized in the P1 fraction (e).

 


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  		Fig. 6. Vid22p co-localizes with the plasma membrane marker protein, Pma1p. The Vid22p-V5 strain was glucose starved and shifted to fresh glucose media for 30 minutes. Cells were lysed and the total lysate was applied to the top of a 20-50% sucrose gradient. Following a 20 hour spin at 100,000 g, fractions from the sucrose gradient were examined for the distribution of Vid22p (a), the plasma membrane marker Pma1p (b), a COP I vesicle protein Sec21p (c), the Golgi marker Mnn1p (d), the vacuolar protein CPY (e) and the Vid vesicle marker protein Vid24p (f).

 


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  		Fig. 7. Vid22p localizes to the plasma membrane. The Vid22p-V5 strain was glucose starved and shifted to fresh glucose media for t=0, 30 and 60 minutes. Immunofluorescence microscopy was then used to determine the localization of Vid22p. Staining of Vid22p appeared to be primarily within the plasma membrane in wild-type cells as determined by FITC-fluorescence labeling (a-c). Pma1p, a plasma membrane marker, exhibited FITC-fluorescence staining on the plasma membrane (d-f). In the {Delta}pep4 strain, the majority of the Vid22p staining was on the plasma membrane either before or after a glucose shift (g-i).

 


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  		Fig. 8. Vid22p is targeted to the plasma membrane independent of the secretory pathway. (A) The sec18-1 mutant strain expressing Vid22p-V5 was radiolabeled for 20 minutes at 37°C and chased for 0, 30 and 60 minutes at 22°C or 37°C. Total lysates were immunoprecipitated with anti-V5 antibodies or anti-CPY antibodies. Immunoprecipitated materials were resolved by SDS-PAGE and radiolabeled proteins were visualized using a phosphorimager. (B) Wild-type cells expressing Vid22p-V5 were labeled for 20 minutes and chased for 0, 30 and 60 minutes at 30°C. Cells were lysed and subjected to differential centrifugation as described in the Materials and Methods section. Each fraction was detergent solubilized and immunoprecipitated with an anti-V5 antibody. The precipitated proteins were solubilized in SDS sample buffer and resolved by SDS-PAGE. Radiolabeled Vid22p was visualized by phosphorimager analysis.

 

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