Limits...
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.

Show MeSH

Related in: MedlinePlus

Differential localization of ALP and other vacuolar  proteins. (A) Double label immunofluorescence of Vph1p (upper  panels) and ALP (middle panels) is shown for wild-type cells  (SF838-9D), vps45Δ cells (RPY12), and vps27Δ cells (HYY1).  The depressions visible in the Nomarski image (lower panels)  identify the yeast vacuole. (B) Immunoprecipitation of newly  synthesized ALP and the soluble vacuolar hydrolase CPY in  wild-type cells (RPY10), vps45Δ cells (RPY11), and vps27Δ cells  (AACY5). Newly synthesized proteins were labeled for 10 min at  30°C with the addition of 35S-Express. Excess unlabeled methio-nine and cysteine were then added for the indicated times. After  centrifugation, internal (pellet) and external (supernatant) fractions were then prepared. CPY was immunoprecipitated from intracellular and extracellular fractions and ALP was immunoprecipitated from intracellular fractions. The PEP4-dependent cleavage  products of ALP are indicated (*).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2141640&req=5

Figure 1: Differential localization of ALP and other vacuolar proteins. (A) Double label immunofluorescence of Vph1p (upper panels) and ALP (middle panels) is shown for wild-type cells (SF838-9D), vps45Δ cells (RPY12), and vps27Δ cells (HYY1). The depressions visible in the Nomarski image (lower panels) identify the yeast vacuole. (B) Immunoprecipitation of newly synthesized ALP and the soluble vacuolar hydrolase CPY in wild-type cells (RPY10), vps45Δ cells (RPY11), and vps27Δ cells (AACY5). Newly synthesized proteins were labeled for 10 min at 30°C with the addition of 35S-Express. Excess unlabeled methio-nine and cysteine were then added for the indicated times. After centrifugation, internal (pellet) and external (supernatant) fractions were then prepared. CPY was immunoprecipitated from intracellular and extracellular fractions and ALP was immunoprecipitated from intracellular fractions. The PEP4-dependent cleavage products of ALP are indicated (*).

Mentions: The recent characterization of many VPS genes has enabled the development of genetic tools that provide specific blocks at various stages along the vacuolar biogenesis pathway from the late Golgi to the vacuole. vps45 mutations appear to block the fusion of a class of Golgi-derived transport vesicles that are destined to fuse with a PVC (Cowles et al., 1994; Piper et al., 1994). This block prevents soluble hydrolases such as CPY and membrane proteins such as Vph1p (an integral membrane subunit of the vacuolar H+-ATPase; Manolson et al., 1992) from reaching the vacuole (Piper et al., 1994). Mutations in VPS27 cause the accumulation of an exaggerated form of a PVC (termed the class E compartment) that traps both Vph1p and CPY (Raymond et al., 1992; Piper et al., 1995). The effect of these blocks can be visualized through the localization of Vph1p in vps45 and vps27 mutants (Fig. 1 A). Wild-type (SF838-9D), vps45Δ (RPY12), and vps27Δ (HYY1) cells were grown at 30°C, fixed, and decorated with anti-Vph1p and anti-ALP antibodies. In contrast with its localization in wild-type cells, Vph1p was not found in the vacuole in vps45Δ cells (defined by Nomarski optics as a large depression). Instead, Vph1p was localized to many small punctate structures consistent with its predicted distribution in the transport vesicles that accumulate in vps45Δ mutants (Cowles et al., 1994; Piper et al., 1995). Similarly, Vph1p was not localized to the vacuole in vps27Δ cells, but instead was found within large perivacuolar class E structures that accumulate when traffic out of the PVC is blocked (Raymond et al., 1992; Piper et al., 1995).


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 localization of ALP and other vacuolar  proteins. (A) Double label immunofluorescence of Vph1p (upper  panels) and ALP (middle panels) is shown for wild-type cells  (SF838-9D), vps45Δ cells (RPY12), and vps27Δ cells (HYY1).  The depressions visible in the Nomarski image (lower panels)  identify the yeast vacuole. (B) Immunoprecipitation of newly  synthesized ALP and the soluble vacuolar hydrolase CPY in  wild-type cells (RPY10), vps45Δ cells (RPY11), and vps27Δ cells  (AACY5). Newly synthesized proteins were labeled for 10 min at  30°C with the addition of 35S-Express. Excess unlabeled methio-nine and cysteine were then added for the indicated times. After  centrifugation, internal (pellet) and external (supernatant) fractions were then prepared. CPY was immunoprecipitated from intracellular and extracellular fractions and ALP was immunoprecipitated from intracellular fractions. The PEP4-dependent cleavage  products of ALP are indicated (*).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141640&req=5

Figure 1: Differential localization of ALP and other vacuolar proteins. (A) Double label immunofluorescence of Vph1p (upper panels) and ALP (middle panels) is shown for wild-type cells (SF838-9D), vps45Δ cells (RPY12), and vps27Δ cells (HYY1). The depressions visible in the Nomarski image (lower panels) identify the yeast vacuole. (B) Immunoprecipitation of newly synthesized ALP and the soluble vacuolar hydrolase CPY in wild-type cells (RPY10), vps45Δ cells (RPY11), and vps27Δ cells (AACY5). Newly synthesized proteins were labeled for 10 min at 30°C with the addition of 35S-Express. Excess unlabeled methio-nine and cysteine were then added for the indicated times. After centrifugation, internal (pellet) and external (supernatant) fractions were then prepared. CPY was immunoprecipitated from intracellular and extracellular fractions and ALP was immunoprecipitated from intracellular fractions. The PEP4-dependent cleavage products of ALP are indicated (*).
Mentions: The recent characterization of many VPS genes has enabled the development of genetic tools that provide specific blocks at various stages along the vacuolar biogenesis pathway from the late Golgi to the vacuole. vps45 mutations appear to block the fusion of a class of Golgi-derived transport vesicles that are destined to fuse with a PVC (Cowles et al., 1994; Piper et al., 1994). This block prevents soluble hydrolases such as CPY and membrane proteins such as Vph1p (an integral membrane subunit of the vacuolar H+-ATPase; Manolson et al., 1992) from reaching the vacuole (Piper et al., 1994). Mutations in VPS27 cause the accumulation of an exaggerated form of a PVC (termed the class E compartment) that traps both Vph1p and CPY (Raymond et al., 1992; Piper et al., 1995). The effect of these blocks can be visualized through the localization of Vph1p in vps45 and vps27 mutants (Fig. 1 A). Wild-type (SF838-9D), vps45Δ (RPY12), and vps27Δ (HYY1) cells were grown at 30°C, fixed, and decorated with anti-Vph1p and anti-ALP antibodies. In contrast with its localization in wild-type cells, Vph1p was not found in the vacuole in vps45Δ cells (defined by Nomarski optics as a large depression). Instead, Vph1p was localized to many small punctate structures consistent with its predicted distribution in the transport vesicles that accumulate in vps45Δ mutants (Cowles et al., 1994; Piper et al., 1995). Similarly, Vph1p was not localized to the vacuole in vps27Δ cells, but instead was found within large perivacuolar class E structures that accumulate when traffic out of the PVC is blocked (Raymond et al., 1992; Piper et al., 1995).

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