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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
Vam3p di-leucine sequence alignment with putative  AP-3 cargoes. The Vam3p di-leucine sequence shares significant  sequence similarity to di-leucine sequences in both lysosomal and  melanosomal proteins. The consensus motif (*Ex*xLL) is derived from mutagenesis data. An asterisk denotes a bias toward  charged, polar amino acids, while x can be any amino acid. Residues that were mutated in the Vam3p sequence and are also conserved in other cargo proteins are indicated by shaded regions.  ALP shares sequence similarity with Vam3p at every base that  was recovered in our Vam3p mutagenesis. The acidic amino acid  at −4, the polar amino acids at −5 and −3, and the proline at −1  as well as the di-leucine sequence, are conserved in the majority  of the proteins that have been defined as potential AP-3 cargoes.
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Figure 6: Vam3p di-leucine sequence alignment with putative AP-3 cargoes. The Vam3p di-leucine sequence shares significant sequence similarity to di-leucine sequences in both lysosomal and melanosomal proteins. The consensus motif (*Ex*xLL) is derived from mutagenesis data. An asterisk denotes a bias toward charged, polar amino acids, while x can be any amino acid. Residues that were mutated in the Vam3p sequence and are also conserved in other cargo proteins are indicated by shaded regions. ALP shares sequence similarity with Vam3p at every base that was recovered in our Vam3p mutagenesis. The acidic amino acid at −4, the polar amino acids at −5 and −3, and the proline at −1 as well as the di-leucine sequence, are conserved in the majority of the proteins that have been defined as potential AP-3 cargoes.

Mentions: Random mutagenesis of Vam3p identified a small 7–amino acid region in the middle of the cytoplasmic domain of Vam3p that includes a leucine pair as well as several amino acids just to the NH2-terminal side of the di-leucine sequence. Because di-leucine sorting signals have been implicated in endocytosis from the plasma membrane (Letourneur and Klausner, 1992; Pond et al., 1995) and also have been shown to be capable of direct binding to both AP-1 and AP-2 in vitro (Heilker et al., 1996; Dietrich et al., 1997; Rapoport et al., 1998), it is likely that only a subset of di-leucine motifs interact with AP-3. Mutations in the glutamate at position −4 relative to the leucine pair in Vam3p resulted in particularly strong phenotypes, comparable to mutations in either of the leucine residues. These results are consistent with in vitro studies that have shown that mutation of the acidic residues in the di-leucine sorting signals in LIMP II and tyrosinase abrogate binding to AP-3–enriched fractions (Honing et al., 1998). However, acidic residues in addition to the di-leucine pair do not seem to be sufficient to direct AP-3 binding, since many mammalian proteins containing similar sequences do not seem to be sorted through AP-3–dependent pathways (Honing et al., 1998). Our analysis has identified several additional residues that also contribute to the function of the di-leucine motif. In addition to the acidic residue at −4 and the leucine pair, the Vam3p motif requires an upstream polar residue at −5 and a hydrophilic amino acid at the −3 position for optimal sorting activity. In fact, the sorting determinant in ALP contains conserved residues at the same positions to each of the additional residues that were mutated in Vam3p (Fig. 6).


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 di-leucine sequence alignment with putative  AP-3 cargoes. The Vam3p di-leucine sequence shares significant  sequence similarity to di-leucine sequences in both lysosomal and  melanosomal proteins. The consensus motif (*Ex*xLL) is derived from mutagenesis data. An asterisk denotes a bias toward  charged, polar amino acids, while x can be any amino acid. Residues that were mutated in the Vam3p sequence and are also conserved in other cargo proteins are indicated by shaded regions.  ALP shares sequence similarity with Vam3p at every base that  was recovered in our Vam3p mutagenesis. The acidic amino acid  at −4, the polar amino acids at −5 and −3, and the proline at −1  as well as the di-leucine sequence, are conserved in the majority  of the proteins that have been defined as potential AP-3 cargoes.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132875&req=5

Figure 6: Vam3p di-leucine sequence alignment with putative AP-3 cargoes. The Vam3p di-leucine sequence shares significant sequence similarity to di-leucine sequences in both lysosomal and melanosomal proteins. The consensus motif (*Ex*xLL) is derived from mutagenesis data. An asterisk denotes a bias toward charged, polar amino acids, while x can be any amino acid. Residues that were mutated in the Vam3p sequence and are also conserved in other cargo proteins are indicated by shaded regions. ALP shares sequence similarity with Vam3p at every base that was recovered in our Vam3p mutagenesis. The acidic amino acid at −4, the polar amino acids at −5 and −3, and the proline at −1 as well as the di-leucine sequence, are conserved in the majority of the proteins that have been defined as potential AP-3 cargoes.
Mentions: Random mutagenesis of Vam3p identified a small 7–amino acid region in the middle of the cytoplasmic domain of Vam3p that includes a leucine pair as well as several amino acids just to the NH2-terminal side of the di-leucine sequence. Because di-leucine sorting signals have been implicated in endocytosis from the plasma membrane (Letourneur and Klausner, 1992; Pond et al., 1995) and also have been shown to be capable of direct binding to both AP-1 and AP-2 in vitro (Heilker et al., 1996; Dietrich et al., 1997; Rapoport et al., 1998), it is likely that only a subset of di-leucine motifs interact with AP-3. Mutations in the glutamate at position −4 relative to the leucine pair in Vam3p resulted in particularly strong phenotypes, comparable to mutations in either of the leucine residues. These results are consistent with in vitro studies that have shown that mutation of the acidic residues in the di-leucine sorting signals in LIMP II and tyrosinase abrogate binding to AP-3–enriched fractions (Honing et al., 1998). However, acidic residues in addition to the di-leucine pair do not seem to be sufficient to direct AP-3 binding, since many mammalian proteins containing similar sequences do not seem to be sorted through AP-3–dependent pathways (Honing et al., 1998). Our analysis has identified several additional residues that also contribute to the function of the di-leucine motif. In addition to the acidic residue at −4 and the leucine pair, the Vam3p motif requires an upstream polar residue at −5 and a hydrophilic amino acid at the −3 position for optimal sorting activity. In fact, the sorting determinant in ALP contains conserved residues at the same positions to each of the additional residues that were mutated in Vam3p (Fig. 6).

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