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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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CPY sorting of  vam3 mutants in both pep12Δ  and vam3Δ mutant cells. (A)  pep12Δ (CBY31) cells and  pep12Δaps3Δ (GOY8) double-mutant cells transformed  with a single-copy plasmid  containing wild-type VAM3  (pVAM3.414), and pep12Δ  (CBY31) cells transformed  with vector (pRS414),  vam3L160P or vam3N154I mutant isolates (pVAM3L160P.  414 and pVAM3N154I.414,  respectively) were spheroplasted, and then metabolically labeled and chased for  45 min. (B) vam3Δ (TDY2)  cells were transformed with  the identical plasmids from A  and were labeled and chased  as whole cells for 30 min. CPY was immunoprecipitated and examined by autoradiography. For both A and B, mature and  Golgi-modified precursor CPY are indicated as mCPY and  p2CPY, respectively. In A, the percent of mature CPY is denoted  beneath each individual lane.
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Figure 3: CPY sorting of vam3 mutants in both pep12Δ and vam3Δ mutant cells. (A) pep12Δ (CBY31) cells and pep12Δaps3Δ (GOY8) double-mutant cells transformed with a single-copy plasmid containing wild-type VAM3 (pVAM3.414), and pep12Δ (CBY31) cells transformed with vector (pRS414), vam3L160P or vam3N154I mutant isolates (pVAM3L160P. 414 and pVAM3N154I.414, respectively) were spheroplasted, and then metabolically labeled and chased for 45 min. (B) vam3Δ (TDY2) cells were transformed with the identical plasmids from A and were labeled and chased as whole cells for 30 min. CPY was immunoprecipitated and examined by autoradiography. For both A and B, mature and Golgi-modified precursor CPY are indicated as mCPY and p2CPY, respectively. In A, the percent of mature CPY is denoted beneath each individual lane.

Mentions: To confirm that the G418 resistance phenotype used to isolate vam3 mutants correlated with suppression of CPY defects of pep12Δ cells harboring these mutant forms of VAM3 we examined CPY sorting by pulse-chase/immunoprecipitation analysis. As expected, in pep12Δ cells CPY was recovered almost exclusively as the Golgi-modified p2 form, consistent with severe defects in Golgi-to-endosome transport. Similarly, pep12Δ cells expressing VAM3 from a single-copy plasmid did not show any significant improvement in CPY sorting. However, in all cases, pep12Δ cells expressing vam3 mutants from a single-copy plasmid that conferred G418 resistance also exhibited improved CPY sorting. Moreover, efficiency of CPY sorting in pep12Δ cells harboring the various vam3 mutants correlated with degree of G418 resistance as mutants that exhibited resistance to high concentrations of G418 sorted and matured more CPY than mutants resistant to only lower (50 μg/ml) G418 concentrations. For example, pep12Δ cells expressing the vam3N154I mutant, which is resistant to G418 concentrations up to 50 μg/ml, converted ∼50% of p2 CPY to the mature form, whereas pep12Δ cells expressing the vam3L160P mutant, which is resistant to 100 μg/ml G418, matured ∼80% of CPY (Fig. 3 A).


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)

CPY sorting of  vam3 mutants in both pep12Δ  and vam3Δ mutant cells. (A)  pep12Δ (CBY31) cells and  pep12Δaps3Δ (GOY8) double-mutant cells transformed  with a single-copy plasmid  containing wild-type VAM3  (pVAM3.414), and pep12Δ  (CBY31) cells transformed  with vector (pRS414),  vam3L160P or vam3N154I mutant isolates (pVAM3L160P.  414 and pVAM3N154I.414,  respectively) were spheroplasted, and then metabolically labeled and chased for  45 min. (B) vam3Δ (TDY2)  cells were transformed with  the identical plasmids from A  and were labeled and chased  as whole cells for 30 min. CPY was immunoprecipitated and examined by autoradiography. For both A and B, mature and  Golgi-modified precursor CPY are indicated as mCPY and  p2CPY, respectively. In A, the percent of mature CPY is denoted  beneath each individual lane.
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Related In: Results  -  Collection

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Figure 3: CPY sorting of vam3 mutants in both pep12Δ and vam3Δ mutant cells. (A) pep12Δ (CBY31) cells and pep12Δaps3Δ (GOY8) double-mutant cells transformed with a single-copy plasmid containing wild-type VAM3 (pVAM3.414), and pep12Δ (CBY31) cells transformed with vector (pRS414), vam3L160P or vam3N154I mutant isolates (pVAM3L160P. 414 and pVAM3N154I.414, respectively) were spheroplasted, and then metabolically labeled and chased for 45 min. (B) vam3Δ (TDY2) cells were transformed with the identical plasmids from A and were labeled and chased as whole cells for 30 min. CPY was immunoprecipitated and examined by autoradiography. For both A and B, mature and Golgi-modified precursor CPY are indicated as mCPY and p2CPY, respectively. In A, the percent of mature CPY is denoted beneath each individual lane.
Mentions: To confirm that the G418 resistance phenotype used to isolate vam3 mutants correlated with suppression of CPY defects of pep12Δ cells harboring these mutant forms of VAM3 we examined CPY sorting by pulse-chase/immunoprecipitation analysis. As expected, in pep12Δ cells CPY was recovered almost exclusively as the Golgi-modified p2 form, consistent with severe defects in Golgi-to-endosome transport. Similarly, pep12Δ cells expressing VAM3 from a single-copy plasmid did not show any significant improvement in CPY sorting. However, in all cases, pep12Δ cells expressing vam3 mutants from a single-copy plasmid that conferred G418 resistance also exhibited improved CPY sorting. Moreover, efficiency of CPY sorting in pep12Δ cells harboring the various vam3 mutants correlated with degree of G418 resistance as mutants that exhibited resistance to high concentrations of G418 sorted and matured more CPY than mutants resistant to only lower (50 μg/ml) G418 concentrations. For example, pep12Δ cells expressing the vam3N154I mutant, which is resistant to G418 concentrations up to 50 μg/ml, converted ∼50% of p2 CPY to the mature form, whereas pep12Δ cells expressing the vam3L160P mutant, which is resistant to 100 μg/ml G418, matured ∼80% of CPY (Fig. 3 A).

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