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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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Trafficking of  ALP and Vps10p-Δ10* in  temperature-sensitive vps  mutants. (A) The rate of  PEP4-dependent processing  of Vps10p-10* (left panels)  and ALP (right panels) was  measured in wild-type cells  (RPY96) and vps45Δ cells  (RPY99) by pulse/chase  analysis. Cells were labeled  with 35S-Express for 10 min  at 30°C and chased for the indicated times. Cell lysates  were divided and subjected  to immunoprecipitation with  anti-ALP and anti-Vps10p  antibodies. Both the full-length Vps10p and the truncated Vps10p-Δ10* were  present. The PEP4-dependent cleavage products of  Vps10p-Δ10* and ALP are  indicated (*). (B) The rate of  PEP4-dependent cleavage  of Vps10p-Δ10* and ALP  was measured in vps45-ts  cells (RPY102). Cells were  grown overnight at 22°C and  then either maintained at  22°C or shifted to 37°C 10  min before labeling with 35S-Express for 10 min. The  chase times used are indicated. ALP and Vps10p-Δ10* were immunoprecipitated as described above. The rate of PEP4-dependent cleavage of Vps10p-Δ10* and ALP was measured in vps27-ts cells (RPY103) and  wild-type cells (RPY110) each carrying the GAL1-PEP4 plasmid, pRCP39. Cells were grown in galactose-containing media for 24 h at  22°C and then shifted to glucose-containing media for 24 h before the pulse/chase immunoprecipitation scheme used for vps45-ts cells  (RPY102) above. Cells were either maintained at 22°C or shifted to 37°C 10 min before labeling.
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Figure 3: Trafficking of ALP and Vps10p-Δ10* in temperature-sensitive vps mutants. (A) The rate of PEP4-dependent processing of Vps10p-10* (left panels) and ALP (right panels) was measured in wild-type cells (RPY96) and vps45Δ cells (RPY99) by pulse/chase analysis. Cells were labeled with 35S-Express for 10 min at 30°C and chased for the indicated times. Cell lysates were divided and subjected to immunoprecipitation with anti-ALP and anti-Vps10p antibodies. Both the full-length Vps10p and the truncated Vps10p-Δ10* were present. The PEP4-dependent cleavage products of Vps10p-Δ10* and ALP are indicated (*). (B) The rate of PEP4-dependent cleavage of Vps10p-Δ10* and ALP was measured in vps45-ts cells (RPY102). Cells were grown overnight at 22°C and then either maintained at 22°C or shifted to 37°C 10 min before labeling with 35S-Express for 10 min. The chase times used are indicated. ALP and Vps10p-Δ10* were immunoprecipitated as described above. The rate of PEP4-dependent cleavage of Vps10p-Δ10* and ALP was measured in vps27-ts cells (RPY103) and wild-type cells (RPY110) each carrying the GAL1-PEP4 plasmid, pRCP39. Cells were grown in galactose-containing media for 24 h at 22°C and then shifted to glucose-containing media for 24 h before the pulse/chase immunoprecipitation scheme used for vps45-ts cells (RPY102) above. Cells were either maintained at 22°C or shifted to 37°C 10 min before labeling.

Mentions: In wild-type cells (RPY96) Vps10p-Δ10* is rapidly processed with a half-time of ∼20 min, consistent with previous measurements (Cooper and Stevens, 1996; Bryant and Stevens, 1997). The full-length Vps10 protein was also followed in these cells to serve as a convenient internal control. In wild-type cells Vps10p is quite stable, owing to tyrosine-based signals within its cytosolic domain that confer its retrieval to the Golgi apparatus and prevent its delivery to the vacuole (Cereghino et al., 1995; Cooper and Stevens, 1996) (Fig. 3). In vps45Δ cells, Vps10p-Δ10* failed to undergo a PEP4-dependent cleavage (Fig. 3 A), consistent with its being trapped in transport vesicles and thus prevented from reaching the vacuole. These data demonstrate that Vps10p-Δ10* can be used to monitor blocks along the VPS-dependent pathway.


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)

Trafficking of  ALP and Vps10p-Δ10* in  temperature-sensitive vps  mutants. (A) The rate of  PEP4-dependent processing  of Vps10p-10* (left panels)  and ALP (right panels) was  measured in wild-type cells  (RPY96) and vps45Δ cells  (RPY99) by pulse/chase  analysis. Cells were labeled  with 35S-Express for 10 min  at 30°C and chased for the indicated times. Cell lysates  were divided and subjected  to immunoprecipitation with  anti-ALP and anti-Vps10p  antibodies. Both the full-length Vps10p and the truncated Vps10p-Δ10* were  present. The PEP4-dependent cleavage products of  Vps10p-Δ10* and ALP are  indicated (*). (B) The rate of  PEP4-dependent cleavage  of Vps10p-Δ10* and ALP  was measured in vps45-ts  cells (RPY102). Cells were  grown overnight at 22°C and  then either maintained at  22°C or shifted to 37°C 10  min before labeling with 35S-Express for 10 min. The  chase times used are indicated. ALP and Vps10p-Δ10* were immunoprecipitated as described above. The rate of PEP4-dependent cleavage of Vps10p-Δ10* and ALP was measured in vps27-ts cells (RPY103) and  wild-type cells (RPY110) each carrying the GAL1-PEP4 plasmid, pRCP39. Cells were grown in galactose-containing media for 24 h at  22°C and then shifted to glucose-containing media for 24 h before the pulse/chase immunoprecipitation scheme used for vps45-ts cells  (RPY102) above. Cells were either maintained at 22°C or shifted to 37°C 10 min before labeling.
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Figure 3: Trafficking of ALP and Vps10p-Δ10* in temperature-sensitive vps mutants. (A) The rate of PEP4-dependent processing of Vps10p-10* (left panels) and ALP (right panels) was measured in wild-type cells (RPY96) and vps45Δ cells (RPY99) by pulse/chase analysis. Cells were labeled with 35S-Express for 10 min at 30°C and chased for the indicated times. Cell lysates were divided and subjected to immunoprecipitation with anti-ALP and anti-Vps10p antibodies. Both the full-length Vps10p and the truncated Vps10p-Δ10* were present. The PEP4-dependent cleavage products of Vps10p-Δ10* and ALP are indicated (*). (B) The rate of PEP4-dependent cleavage of Vps10p-Δ10* and ALP was measured in vps45-ts cells (RPY102). Cells were grown overnight at 22°C and then either maintained at 22°C or shifted to 37°C 10 min before labeling with 35S-Express for 10 min. The chase times used are indicated. ALP and Vps10p-Δ10* were immunoprecipitated as described above. The rate of PEP4-dependent cleavage of Vps10p-Δ10* and ALP was measured in vps27-ts cells (RPY103) and wild-type cells (RPY110) each carrying the GAL1-PEP4 plasmid, pRCP39. Cells were grown in galactose-containing media for 24 h at 22°C and then shifted to glucose-containing media for 24 h before the pulse/chase immunoprecipitation scheme used for vps45-ts cells (RPY102) above. Cells were either maintained at 22°C or shifted to 37°C 10 min before labeling.
Mentions: In wild-type cells (RPY96) Vps10p-Δ10* is rapidly processed with a half-time of ∼20 min, consistent with previous measurements (Cooper and Stevens, 1996; Bryant and Stevens, 1997). The full-length Vps10 protein was also followed in these cells to serve as a convenient internal control. In wild-type cells Vps10p is quite stable, owing to tyrosine-based signals within its cytosolic domain that confer its retrieval to the Golgi apparatus and prevent its delivery to the vacuole (Cereghino et al., 1995; Cooper and Stevens, 1996) (Fig. 3). In vps45Δ cells, Vps10p-Δ10* failed to undergo a PEP4-dependent cleavage (Fig. 3 A), consistent with its being trapped in transport vesicles and thus prevented from reaching the vacuole. These data demonstrate that Vps10p-Δ10* can be used to monitor blocks along the VPS-dependent pathway.

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