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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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Vam3p mutants define a di-leucine sorting signal.  Vam3 protein domain structure is shown including the extreme  COOH-terminal transmembrane domain and the two coiled-coil  domains. The location of the region containing the mutations recovered from the G418 resistance screen is denoted by the  dashed box. The wild-type sequence at the mutated region is  shown in detail. Single mutations that were recovered from the  G418 resistance screen are indicated in capital letters designating  the amino acid changes. The glutamine 156 to leucine mutation  was not recovered in the screen but was made by site-directed  mutagenesis. The level of G418 resistance conferred by each of  the mutants is as follows: ++, wild-type growth; +, slower  growth to single colonies; and −, no growth.
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Figure 2: Vam3p mutants define a di-leucine sorting signal. Vam3 protein domain structure is shown including the extreme COOH-terminal transmembrane domain and the two coiled-coil domains. The location of the region containing the mutations recovered from the G418 resistance screen is denoted by the dashed box. The wild-type sequence at the mutated region is shown in detail. Single mutations that were recovered from the G418 resistance screen are indicated in capital letters designating the amino acid changes. The glutamine 156 to leucine mutation was not recovered in the screen but was made by site-directed mutagenesis. The level of G418 resistance conferred by each of the mutants is as follows: ++, wild-type growth; +, slower growth to single colonies; and −, no growth.

Mentions: Sequence analysis revealed that each of the 30 mutants recovered in this screen contained at least one mutation within a 7–amino acid region of Vam3p, corresponding to amino acids 154–160 (Fig. 2). This sequence of Vam3p lies approximately in the middle of the protein, between the two coiled-coil domains, and it is distanced well away from the transmembrane domain in a region that does not exhibit high sequence similarity to Pep12p. However, the sequence closely resembles a region in the cytosolic tail of ALP that has been shown to be necessary for transport of ALP through the AP-3–dependent pathway to the vacuole (Cowles et al., 1997b; Vowels and Payne, 1998). These characteristics, together with the presence of a leucine pair at positions 159–160, suggested that this region may represent a di-leucine–type sorting signal in Vam3p.


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)

Vam3p mutants define a di-leucine sorting signal.  Vam3 protein domain structure is shown including the extreme  COOH-terminal transmembrane domain and the two coiled-coil  domains. The location of the region containing the mutations recovered from the G418 resistance screen is denoted by the  dashed box. The wild-type sequence at the mutated region is  shown in detail. Single mutations that were recovered from the  G418 resistance screen are indicated in capital letters designating  the amino acid changes. The glutamine 156 to leucine mutation  was not recovered in the screen but was made by site-directed  mutagenesis. The level of G418 resistance conferred by each of  the mutants is as follows: ++, wild-type growth; +, slower  growth to single colonies; and −, no growth.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Vam3p mutants define a di-leucine sorting signal. Vam3 protein domain structure is shown including the extreme COOH-terminal transmembrane domain and the two coiled-coil domains. The location of the region containing the mutations recovered from the G418 resistance screen is denoted by the dashed box. The wild-type sequence at the mutated region is shown in detail. Single mutations that were recovered from the G418 resistance screen are indicated in capital letters designating the amino acid changes. The glutamine 156 to leucine mutation was not recovered in the screen but was made by site-directed mutagenesis. The level of G418 resistance conferred by each of the mutants is as follows: ++, wild-type growth; +, slower growth to single colonies; and −, no growth.
Mentions: Sequence analysis revealed that each of the 30 mutants recovered in this screen contained at least one mutation within a 7–amino acid region of Vam3p, corresponding to amino acids 154–160 (Fig. 2). This sequence of Vam3p lies approximately in the middle of the protein, between the two coiled-coil domains, and it is distanced well away from the transmembrane domain in a region that does not exhibit high sequence similarity to Pep12p. However, the sequence closely resembles a region in the cytosolic tail of ALP that has been shown to be necessary for transport of ALP through the AP-3–dependent pathway to the vacuole (Cowles et al., 1997b; Vowels and Payne, 1998). These characteristics, together with the presence of a leucine pair at positions 159–160, suggested that this region may represent a di-leucine–type sorting signal in Vam3p.

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