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Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p.

Nothwehr SF, Ha SA, Bruinsma P - J. Cell Biol. (2000)

Bottom Line: By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins.Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 mutation.Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211, USA. nothwehrs@missouri.edu

ABSTRACT
Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar-endosomal compartment, a process dependent on aromatic amino acid-based sorting determinants on their cytosolic domains. The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex. We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval. By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins. Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal-dependent manner. Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 mutation. Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment. These data strongly support a model in which Vps35p acts as a "receptor" protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles.

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The Vps10-Q1499L mutant protein exhibits a predominantly Golgi localization pattern in vps35-105 cells. The following strains (shown respectively from left to right) were analyzed: SNY135, SNY132, SNY136, SNY133, and SNY137. Strains propagated at 30°C were fixed, spheroplasted, and costained with antibodies against the HA epitope and Vma2p. After subsequent detection with fluorochromes (see Materials and Methods) the cells were viewed by DIC optics and by epifluorescence through filters specific for FITC and Texas red.
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Figure 6: The Vps10-Q1499L mutant protein exhibits a predominantly Golgi localization pattern in vps35-105 cells. The following strains (shown respectively from left to right) were analyzed: SNY135, SNY132, SNY136, SNY133, and SNY137. Strains propagated at 30°C were fixed, spheroplasted, and costained with antibodies against the HA epitope and Vma2p. After subsequent detection with fluorochromes (see Materials and Methods) the cells were viewed by DIC optics and by epifluorescence through filters specific for FITC and Texas red.

Mentions: Localization of wild-type and mutant Vps10p was also analyzed by immunofluorescence microscopy. To facilitate detection, three copies of the HA epitope tag were fused to the COOH terminus of wild-type and mutant Vps10p. COOH-terminal addition of the HA tag does not appear to affect the function or trafficking of Vps10p since VPS10::HA strains exhibit normal CPY sorting (data not shown). The Vps10-Q1499L protein in the VPS35 vps10-Q1499L::HA strain exhibits a punctate staining pattern (Fig. 6) typical of TGN resident proteins. In contrast, both wild-type and mutant Vps10 proteins exhibit striking vacuolar localization in vps35Δ cells. Wild-type Vps10p is also clearly mislocalized to the vacuole in the vps35-105 background. However, the suppressor Vps10-Q1499L mutant in the vps35-105 background exhibits a mostly punctate Golgi-like staining pattern along with a minor amount of vacuolar membrane staining (compare the anti-HA and anti-Vma2p panels).


Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p.

Nothwehr SF, Ha SA, Bruinsma P - J. Cell Biol. (2000)

The Vps10-Q1499L mutant protein exhibits a predominantly Golgi localization pattern in vps35-105 cells. The following strains (shown respectively from left to right) were analyzed: SNY135, SNY132, SNY136, SNY133, and SNY137. Strains propagated at 30°C were fixed, spheroplasted, and costained with antibodies against the HA epitope and Vma2p. After subsequent detection with fluorochromes (see Materials and Methods) the cells were viewed by DIC optics and by epifluorescence through filters specific for FITC and Texas red.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192648&req=5

Figure 6: The Vps10-Q1499L mutant protein exhibits a predominantly Golgi localization pattern in vps35-105 cells. The following strains (shown respectively from left to right) were analyzed: SNY135, SNY132, SNY136, SNY133, and SNY137. Strains propagated at 30°C were fixed, spheroplasted, and costained with antibodies against the HA epitope and Vma2p. After subsequent detection with fluorochromes (see Materials and Methods) the cells were viewed by DIC optics and by epifluorescence through filters specific for FITC and Texas red.
Mentions: Localization of wild-type and mutant Vps10p was also analyzed by immunofluorescence microscopy. To facilitate detection, three copies of the HA epitope tag were fused to the COOH terminus of wild-type and mutant Vps10p. COOH-terminal addition of the HA tag does not appear to affect the function or trafficking of Vps10p since VPS10::HA strains exhibit normal CPY sorting (data not shown). The Vps10-Q1499L protein in the VPS35 vps10-Q1499L::HA strain exhibits a punctate staining pattern (Fig. 6) typical of TGN resident proteins. In contrast, both wild-type and mutant Vps10 proteins exhibit striking vacuolar localization in vps35Δ cells. Wild-type Vps10p is also clearly mislocalized to the vacuole in the vps35-105 background. However, the suppressor Vps10-Q1499L mutant in the vps35-105 background exhibits a mostly punctate Golgi-like staining pattern along with a minor amount of vacuolar membrane staining (compare the anti-HA and anti-Vma2p panels).

Bottom Line: By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins.Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 mutation.Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211, USA. nothwehrs@missouri.edu

ABSTRACT
Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar-endosomal compartment, a process dependent on aromatic amino acid-based sorting determinants on their cytosolic domains. The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex. We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval. By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins. Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal-dependent manner. Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 mutation. Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment. These data strongly support a model in which Vps35p acts as a "receptor" protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles.

Show MeSH
Related in: MedlinePlus