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The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway.

Piper RC, Bryant NJ, Stevens TH - J. Cell Biol. (1997)

Bottom Line: Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.

ABSTRACT
Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment. The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole. Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

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Differential targeting of ALP and Vph1p in temperature-sensitive vps mutants. The targeting of newly synthesized  Vph1p and ALP was monitored in vps45-ts cells (RPY94; top  panels), vps27-ts cells (RPY90; middle panels), and wild-type cells  (RPY111; bottom panels). The production of both ALP and  Vph1p in RPY94, RPY90, and RPY111 cells was under the control of the galactose-inducible GAL1 promoter. Cells were grown  overnight at 22°C in the absence of galactose. Galactose was then  added, and cells were incubated for 45 min at 22°C or immediately shifted to 37°C as indicated. Cycloheximide was then added  (100 μg/ml) for an additional 10 min before fixation and double  labeled for Vph1p and ALP using indirect immunofluorescence.  Vph1p was labeled with rabbit anti-Vph1p antibodies and Texas  red–conjugated secondary antibody (left panels); ALP was labeled with anti-ALP 1D3 mAb with biotinylated secondary and  FITC-conjugated streptavidin. Shown as a control are wild-type  cells (RPY111) incubated at 37°C in the presence or absence of  galactose (lower panels).
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Figure 2: Differential targeting of ALP and Vph1p in temperature-sensitive vps mutants. The targeting of newly synthesized Vph1p and ALP was monitored in vps45-ts cells (RPY94; top panels), vps27-ts cells (RPY90; middle panels), and wild-type cells (RPY111; bottom panels). The production of both ALP and Vph1p in RPY94, RPY90, and RPY111 cells was under the control of the galactose-inducible GAL1 promoter. Cells were grown overnight at 22°C in the absence of galactose. Galactose was then added, and cells were incubated for 45 min at 22°C or immediately shifted to 37°C as indicated. Cycloheximide was then added (100 μg/ml) for an additional 10 min before fixation and double labeled for Vph1p and ALP using indirect immunofluorescence. Vph1p was labeled with rabbit anti-Vph1p antibodies and Texas red–conjugated secondary antibody (left panels); ALP was labeled with anti-ALP 1D3 mAb with biotinylated secondary and FITC-conjugated streptavidin. Shown as a control are wild-type cells (RPY111) incubated at 37°C in the presence or absence of galactose (lower panels).

Mentions: To address these concerns, we used cells carrying temperature-sensitive alleles of either VPS45 or VPS27 that rapidly lose their function in cells shifted from 22°C to 37°C. This strategy ensured that the vacuolar biogenesis pathway(s) suffered no long-term defects and that vacuolar morphology and function were normal. We then followed a wave of newly synthesized ALP and Vph1p using immunofluorescence localization in cells that had undergone rapid inactivation of Vps45p or Vps27p function (Fig. 2). For these experiments, the VPH1 and PHO8 (ALP) open reading frames were placed under the control of the inducible GAL1 promoter. This was accomplished by creating a tandem integration at both the VPH1 and PHO8 loci, which truncated the endogenous copy of the gene while inserting a new copy of the gene containing the GAL1 promoter. Within 40 min of adding galactose, the level of induced protein was sufficient to follow newly synthesized protein by immunofluorescence localization as shown in Fig. 2 (bottom) where ALP and Vph1p labeling were only discernible in cells induced by galactose.


The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway.

Piper RC, Bryant NJ, Stevens TH - J. Cell Biol. (1997)

Differential targeting of ALP and Vph1p in temperature-sensitive vps mutants. The targeting of newly synthesized  Vph1p and ALP was monitored in vps45-ts cells (RPY94; top  panels), vps27-ts cells (RPY90; middle panels), and wild-type cells  (RPY111; bottom panels). The production of both ALP and  Vph1p in RPY94, RPY90, and RPY111 cells was under the control of the galactose-inducible GAL1 promoter. Cells were grown  overnight at 22°C in the absence of galactose. Galactose was then  added, and cells were incubated for 45 min at 22°C or immediately shifted to 37°C as indicated. Cycloheximide was then added  (100 μg/ml) for an additional 10 min before fixation and double  labeled for Vph1p and ALP using indirect immunofluorescence.  Vph1p was labeled with rabbit anti-Vph1p antibodies and Texas  red–conjugated secondary antibody (left panels); ALP was labeled with anti-ALP 1D3 mAb with biotinylated secondary and  FITC-conjugated streptavidin. Shown as a control are wild-type  cells (RPY111) incubated at 37°C in the presence or absence of  galactose (lower panels).
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Figure 2: Differential targeting of ALP and Vph1p in temperature-sensitive vps mutants. The targeting of newly synthesized Vph1p and ALP was monitored in vps45-ts cells (RPY94; top panels), vps27-ts cells (RPY90; middle panels), and wild-type cells (RPY111; bottom panels). The production of both ALP and Vph1p in RPY94, RPY90, and RPY111 cells was under the control of the galactose-inducible GAL1 promoter. Cells were grown overnight at 22°C in the absence of galactose. Galactose was then added, and cells were incubated for 45 min at 22°C or immediately shifted to 37°C as indicated. Cycloheximide was then added (100 μg/ml) for an additional 10 min before fixation and double labeled for Vph1p and ALP using indirect immunofluorescence. Vph1p was labeled with rabbit anti-Vph1p antibodies and Texas red–conjugated secondary antibody (left panels); ALP was labeled with anti-ALP 1D3 mAb with biotinylated secondary and FITC-conjugated streptavidin. Shown as a control are wild-type cells (RPY111) incubated at 37°C in the presence or absence of galactose (lower panels).
Mentions: To address these concerns, we used cells carrying temperature-sensitive alleles of either VPS45 or VPS27 that rapidly lose their function in cells shifted from 22°C to 37°C. This strategy ensured that the vacuolar biogenesis pathway(s) suffered no long-term defects and that vacuolar morphology and function were normal. We then followed a wave of newly synthesized ALP and Vph1p using immunofluorescence localization in cells that had undergone rapid inactivation of Vps45p or Vps27p function (Fig. 2). For these experiments, the VPH1 and PHO8 (ALP) open reading frames were placed under the control of the inducible GAL1 promoter. This was accomplished by creating a tandem integration at both the VPH1 and PHO8 loci, which truncated the endogenous copy of the gene while inserting a new copy of the gene containing the GAL1 promoter. Within 40 min of adding galactose, the level of induced protein was sufficient to follow newly synthesized protein by immunofluorescence localization as shown in Fig. 2 (bottom) where ALP and Vph1p labeling were only discernible in cells induced by galactose.

Bottom Line: Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.

ABSTRACT
Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment. The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole. Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

Show MeSH
Related in: MedlinePlus