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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.

Show MeSH
Novel genetic screen to identify regions of Vam3p required for function and localization at the vacuole. (A) In wild-type cells, Pep12p, the endosomal t-SNARE, directs traffic from  the Golgi complex to the endosome through the CPY pathway.  Vam3p, the vacuolar t-SNARE, is transported through the AP-3–dependent ALP pathway to the vacuole where it functions to  receive incoming vesicular traffic from both the ALP and CPY  pathways. We designed a G418-based selection scheme to identify  VAM3 mutants that were able to compensate for the loss of  PEP12 and thus identify regions of Vam3p sequence responsible  for restricting Vam3p activity to the AP-3–dependent pathway.  (B) Growth of both pep12Δ (CBY31) cells alone or pep12Δ  (CBY31) cells expressing VAM3 from a single-copy plasmid  (pVAM3.414) is compromised at concentrations of 50 μg/ml  G418, while growth on media containing no G418 is normal. However, pep12Δ (CBY31) cells either overexpressing VAM3 from a  2μ plasmid (pVAM3.424) or expressing a mutant derived from  the screen, vam3L160P, on a single-copy plasmid (pVAM3L160P.414)  results in growth at 50 μg/ml G418, as well as on media containing  no G418.
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Figure 1: Novel genetic screen to identify regions of Vam3p required for function and localization at the vacuole. (A) In wild-type cells, Pep12p, the endosomal t-SNARE, directs traffic from the Golgi complex to the endosome through the CPY pathway. Vam3p, the vacuolar t-SNARE, is transported through the AP-3–dependent ALP pathway to the vacuole where it functions to receive incoming vesicular traffic from both the ALP and CPY pathways. We designed a G418-based selection scheme to identify VAM3 mutants that were able to compensate for the loss of PEP12 and thus identify regions of Vam3p sequence responsible for restricting Vam3p activity to the AP-3–dependent pathway. (B) Growth of both pep12Δ (CBY31) cells alone or pep12Δ (CBY31) cells expressing VAM3 from a single-copy plasmid (pVAM3.414) is compromised at concentrations of 50 μg/ml G418, while growth on media containing no G418 is normal. However, pep12Δ (CBY31) cells either overexpressing VAM3 from a 2μ plasmid (pVAM3.424) or expressing a mutant derived from the screen, vam3L160P, on a single-copy plasmid (pVAM3L160P.414) results in growth at 50 μg/ml G418, as well as on media containing no G418.

Mentions: Vam3p and Pep12p are two homologous yeast t-SNARE proteins involved in protein transport to the vacuole in yeast. Although Vam3p and Pep12p have clearly distinguishable functions (i.e., Pep12p acts at the endosome and Vam3p acts at the vacuole) (Fig. 1 A), when overexpressed, these proteins are able to partially substitute for one another (Darsow et al., 1997; Gotte and Gallwitz, 1997). This suggests that the specificity of Vam3p and Pep12p functions may be dependent on interactions with other compartment-specific transport components (Sec1 homologues, Rab proteins). Based on this observation, we reasoned that mutations in Vam3p that allow it to substitute for Pep12p might uncover domains required for localization and/or specific protein–protein interactions of Vam3p with Pep12p-specific transport components.


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)

Novel genetic screen to identify regions of Vam3p required for function and localization at the vacuole. (A) In wild-type cells, Pep12p, the endosomal t-SNARE, directs traffic from  the Golgi complex to the endosome through the CPY pathway.  Vam3p, the vacuolar t-SNARE, is transported through the AP-3–dependent ALP pathway to the vacuole where it functions to  receive incoming vesicular traffic from both the ALP and CPY  pathways. We designed a G418-based selection scheme to identify  VAM3 mutants that were able to compensate for the loss of  PEP12 and thus identify regions of Vam3p sequence responsible  for restricting Vam3p activity to the AP-3–dependent pathway.  (B) Growth of both pep12Δ (CBY31) cells alone or pep12Δ  (CBY31) cells expressing VAM3 from a single-copy plasmid  (pVAM3.414) is compromised at concentrations of 50 μg/ml  G418, while growth on media containing no G418 is normal. However, pep12Δ (CBY31) cells either overexpressing VAM3 from a  2μ plasmid (pVAM3.424) or expressing a mutant derived from  the screen, vam3L160P, on a single-copy plasmid (pVAM3L160P.414)  results in growth at 50 μg/ml G418, as well as on media containing  no G418.
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Related In: Results  -  Collection

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Figure 1: Novel genetic screen to identify regions of Vam3p required for function and localization at the vacuole. (A) In wild-type cells, Pep12p, the endosomal t-SNARE, directs traffic from the Golgi complex to the endosome through the CPY pathway. Vam3p, the vacuolar t-SNARE, is transported through the AP-3–dependent ALP pathway to the vacuole where it functions to receive incoming vesicular traffic from both the ALP and CPY pathways. We designed a G418-based selection scheme to identify VAM3 mutants that were able to compensate for the loss of PEP12 and thus identify regions of Vam3p sequence responsible for restricting Vam3p activity to the AP-3–dependent pathway. (B) Growth of both pep12Δ (CBY31) cells alone or pep12Δ (CBY31) cells expressing VAM3 from a single-copy plasmid (pVAM3.414) is compromised at concentrations of 50 μg/ml G418, while growth on media containing no G418 is normal. However, pep12Δ (CBY31) cells either overexpressing VAM3 from a 2μ plasmid (pVAM3.424) or expressing a mutant derived from the screen, vam3L160P, on a single-copy plasmid (pVAM3L160P.414) results in growth at 50 μg/ml G418, as well as on media containing no G418.
Mentions: Vam3p and Pep12p are two homologous yeast t-SNARE proteins involved in protein transport to the vacuole in yeast. Although Vam3p and Pep12p have clearly distinguishable functions (i.e., Pep12p acts at the endosome and Vam3p acts at the vacuole) (Fig. 1 A), when overexpressed, these proteins are able to partially substitute for one another (Darsow et al., 1997; Gotte and Gallwitz, 1997). This suggests that the specificity of Vam3p and Pep12p functions may be dependent on interactions with other compartment-specific transport components (Sec1 homologues, Rab proteins). Based on this observation, we reasoned that mutations in Vam3p that allow it to substitute for Pep12p might uncover domains required for localization and/or specific protein–protein interactions of Vam3p with Pep12p-specific transport components.

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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