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Acidic di-leucine motif essential for AP-3-dependent sorting and restriction of the functional specificity of the Vam3p vacuolar t-SNARE.

Darsow T, Burd CG, Emr SD - J. Cell Biol. (1998)

Bottom Line: Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal.Together, these data provide the first identification of an adaptor protein-specific sorting signal in a t-SNARE protein, and suggest that AP-3-dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function.Regulated transport of cargoes such as Vam3p through the AP-3-dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668, USA. semr@ucsd.edu

ABSTRACT
The transport of newly synthesized proteins through the vacuolar protein sorting pathway in the budding yeast Saccharomyces cerevisiae requires two distinct target SNAP receptor (t-SNARE) proteins, Pep12p and Vam3p. Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport of proteins from the Golgi to the vacuole through a well defined route, the carboxypeptidase Y (CPY) pathway. Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently described alkaline phosphatase (ALP) pathways. Surprisingly, despite their organelle-specific functions in sorting of vacuolar proteins, overexpression of VAM3 can suppress the protein sorting defects of pep12Delta cells. Based on this observation, we developed a genetic screen to identify domains in Vam3p (e.g., localization and/or specific protein-protein interaction domains) that allow it to efficiently substitute for Pep12p. Using this screen, we identified mutations in a 7-amino acid sequence in Vam3p that lead to missorting of Vam3p from the ALP pathway into the CPY pathway where it can substitute for Pep12p at the pre-vacuolar endosome. This region contains an acidic di-leucine sequence that is closely related to sorting signals required for AP-3 adaptor-dependent transport in both yeast and mammalian systems. Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal. Together, these data provide the first identification of an adaptor protein-specific sorting signal in a t-SNARE protein, and suggest that AP-3-dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function. Regulated transport of cargoes such as Vam3p through the AP-3-dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.

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Localization of Vam3 mutant proteins in vps24Δ cells.  vam3Δ (TDY2) and vps24Δ (BW102) cells were transformed  with plasmids containing both wild-type (pGFPVAM3.426) and  mutant (pGFPVAM3L160P.426) GFP-Vam3 fusion proteins.  These strains were grown in selective media to exponential  phase, harvested, and then resuspended in YNB for examination  by microscopy. vps24Δ cultures were further labeled with 16 μM  FM4-64 for a period of 1 h at 26°C in YPD. Labeled cells were  then harvested, resuspended in YPD, and then chased for 1.5 h.  After chase, cells were examined by Nomarski and fluorescence/ confocal microscopy for both GFP and FM4-64 fluorescence. Arrows in A indicate vacuoles and in B indicate class E compartments.
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Figure 5: Localization of Vam3 mutant proteins in vps24Δ cells. vam3Δ (TDY2) and vps24Δ (BW102) cells were transformed with plasmids containing both wild-type (pGFPVAM3.426) and mutant (pGFPVAM3L160P.426) GFP-Vam3 fusion proteins. These strains were grown in selective media to exponential phase, harvested, and then resuspended in YNB for examination by microscopy. vps24Δ cultures were further labeled with 16 μM FM4-64 for a period of 1 h at 26°C in YPD. Labeled cells were then harvested, resuspended in YPD, and then chased for 1.5 h. After chase, cells were examined by Nomarski and fluorescence/ confocal microscopy for both GFP and FM4-64 fluorescence. Arrows in A indicate vacuoles and in B indicate class E compartments.

Mentions: The cell fractionation data suggested that a significant portion of the mutant Vam3 proteins were mislocalized to compartments other than the vacuole. To visualize the compartments in which the mislocalized proteins were residing, we constructed GFP fusions with wild-type and mutant Vam3 proteins. We transformed the plasmid constructs into vam3Δ cells and examined the cells by fluorescence microscopy. Both the wild-type and mutant Vam3 fusion proteins complemented the protein sorting (data not shown) and morphology defects associated with vam3Δ cells (Fig. 5 A) and thus functioned as the native proteins. Consistent with the previous localization studies, wild-type GFP-Vam3 fusion protein localized almost exclusively to vacuolar membranes (Fig. 5 A). While GFP-Vam3L160P still accumulated in vacuolar membranes, a large portion of the protein was also seen in tubular and punctate structures throughout the cell (Fig. 5 A). GFP fusion proteins of the other Vam3p mutants also behaved in a similar manner (data not shown), indicating that these mutant proteins accumulate in non-vacuolar structures.


Acidic di-leucine motif essential for AP-3-dependent sorting and restriction of the functional specificity of the Vam3p vacuolar t-SNARE.

Darsow T, Burd CG, Emr SD - J. Cell Biol. (1998)

Localization of Vam3 mutant proteins in vps24Δ cells.  vam3Δ (TDY2) and vps24Δ (BW102) cells were transformed  with plasmids containing both wild-type (pGFPVAM3.426) and  mutant (pGFPVAM3L160P.426) GFP-Vam3 fusion proteins.  These strains were grown in selective media to exponential  phase, harvested, and then resuspended in YNB for examination  by microscopy. vps24Δ cultures were further labeled with 16 μM  FM4-64 for a period of 1 h at 26°C in YPD. Labeled cells were  then harvested, resuspended in YPD, and then chased for 1.5 h.  After chase, cells were examined by Nomarski and fluorescence/ confocal microscopy for both GFP and FM4-64 fluorescence. Arrows in A indicate vacuoles and in B indicate class E compartments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132875&req=5

Figure 5: Localization of Vam3 mutant proteins in vps24Δ cells. vam3Δ (TDY2) and vps24Δ (BW102) cells were transformed with plasmids containing both wild-type (pGFPVAM3.426) and mutant (pGFPVAM3L160P.426) GFP-Vam3 fusion proteins. These strains were grown in selective media to exponential phase, harvested, and then resuspended in YNB for examination by microscopy. vps24Δ cultures were further labeled with 16 μM FM4-64 for a period of 1 h at 26°C in YPD. Labeled cells were then harvested, resuspended in YPD, and then chased for 1.5 h. After chase, cells were examined by Nomarski and fluorescence/ confocal microscopy for both GFP and FM4-64 fluorescence. Arrows in A indicate vacuoles and in B indicate class E compartments.
Mentions: The cell fractionation data suggested that a significant portion of the mutant Vam3 proteins were mislocalized to compartments other than the vacuole. To visualize the compartments in which the mislocalized proteins were residing, we constructed GFP fusions with wild-type and mutant Vam3 proteins. We transformed the plasmid constructs into vam3Δ cells and examined the cells by fluorescence microscopy. Both the wild-type and mutant Vam3 fusion proteins complemented the protein sorting (data not shown) and morphology defects associated with vam3Δ cells (Fig. 5 A) and thus functioned as the native proteins. Consistent with the previous localization studies, wild-type GFP-Vam3 fusion protein localized almost exclusively to vacuolar membranes (Fig. 5 A). While GFP-Vam3L160P still accumulated in vacuolar membranes, a large portion of the protein was also seen in tubular and punctate structures throughout the cell (Fig. 5 A). GFP fusion proteins of the other Vam3p mutants also behaved in a similar manner (data not shown), indicating that these mutant proteins accumulate in non-vacuolar structures.

Bottom Line: Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal.Together, these data provide the first identification of an adaptor protein-specific sorting signal in a t-SNARE protein, and suggest that AP-3-dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function.Regulated transport of cargoes such as Vam3p through the AP-3-dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular and Molecular Medicine and Department of Biology, Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668, USA. semr@ucsd.edu

ABSTRACT
The transport of newly synthesized proteins through the vacuolar protein sorting pathway in the budding yeast Saccharomyces cerevisiae requires two distinct target SNAP receptor (t-SNARE) proteins, Pep12p and Vam3p. Pep12p is localized to the pre-vacuolar endosome and its activity is required for transport of proteins from the Golgi to the vacuole through a well defined route, the carboxypeptidase Y (CPY) pathway. Vam3p is localized to the vacuole where it mediates delivery of cargoes from both the CPY and the recently described alkaline phosphatase (ALP) pathways. Surprisingly, despite their organelle-specific functions in sorting of vacuolar proteins, overexpression of VAM3 can suppress the protein sorting defects of pep12Delta cells. Based on this observation, we developed a genetic screen to identify domains in Vam3p (e.g., localization and/or specific protein-protein interaction domains) that allow it to efficiently substitute for Pep12p. Using this screen, we identified mutations in a 7-amino acid sequence in Vam3p that lead to missorting of Vam3p from the ALP pathway into the CPY pathway where it can substitute for Pep12p at the pre-vacuolar endosome. This region contains an acidic di-leucine sequence that is closely related to sorting signals required for AP-3 adaptor-dependent transport in both yeast and mammalian systems. Furthermore, disruption of AP-3 function also results in the ability of wild-type Vam3p to compensate for pep12 mutants, suggesting that AP-3 mediates the sorting of Vam3p via the di-leucine signal. Together, these data provide the first identification of an adaptor protein-specific sorting signal in a t-SNARE protein, and suggest that AP-3-dependent sorting of Vam3p acts to restrict its interaction with compartment-specific accessory proteins, thereby regulating its function. Regulated transport of cargoes such as Vam3p through the AP-3-dependent pathway may play an important role in maintaining the unique composition, function, and morphology of the vacuole.

Show MeSH
Related in: MedlinePlus