Limits...
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
Localization of  Vam3 mutant proteins.  Cleared cell lysates generated  from vam3Δ (TDY2) cells  containing either wild-type  VAM3 (pVAM3.414) or the  vam3L160P mutant (pVAM3L160P.414) were loaded onto  the top of an Accudenz step  gradient and centrifuged to  equilibrium. Fractions 1–12  were collected from the top  of the gradient. Proteins  were precipitated from the  fractions and then separated  by SDS-PAGE and transferred to nitrocellulose.  Vam3p, Vph1p, and Pep12p  were detected by immunoblotting and visualized by  ECL fluorography. The distribution of both wild-type and  mutant (Vam3pL160P) Vam3p  and Vph1p are shown graphically in A and the colocalization of Vam3pL160P and  Pep12p are shown in B.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132875&req=5

Figure 4: Localization of Vam3 mutant proteins. Cleared cell lysates generated from vam3Δ (TDY2) cells containing either wild-type VAM3 (pVAM3.414) or the vam3L160P mutant (pVAM3L160P.414) were loaded onto the top of an Accudenz step gradient and centrifuged to equilibrium. Fractions 1–12 were collected from the top of the gradient. Proteins were precipitated from the fractions and then separated by SDS-PAGE and transferred to nitrocellulose. Vam3p, Vph1p, and Pep12p were detected by immunoblotting and visualized by ECL fluorography. The distribution of both wild-type and mutant (Vam3pL160P) Vam3p and Vph1p are shown graphically in A and the colocalization of Vam3pL160P and Pep12p are shown in B.

Mentions: The ability of the vam3 mutants to suppress pep12Δ vacuolar protein sorting defects suggests that mutant Vam3 proteins are at least partially localized to the pre-vacuolar endosome, the site of Pep12p function. To examine the localization of the Vam3 mutant proteins, we fractionated cells and examined the distribution of the Vam3 mutant proteins relative to wild-type Vam3p by differential centrifugation. Vam3p fractionates exclusively in a low speed (P13) pellet fraction by differential centrifugation, which is consistent with its vacuolar localization (Darsow et al., 1997). However, the mutant forms of Vam3p fractionated differently than the wild-type protein, with a significant portion of the proteins localized to a high speed P100 pellet, (data not shown) which indicated that at least a portion of the mutant proteins localized to a non-vacuolar fraction. Although the P13 fraction is enriched in vacuoles, other compartments also fractionate in the P13. For example, the endosomal t-SNARE Pep12p typically exhibits a 40% P13/60% P100 fractionation pattern, even though this protein is not localized to the vacuole (Becherer et al., 1996). To further resolve vacuoles away from other P13 material, vam3Δ cells harboring either single-copy wild-type VAM3 or the vam3L160P mutant were analyzed by equilibrium density gradient fractionation. Cleared spheroplast lysates were applied to the top of Accudenz step gradients, which were then centrifuged to equilibrium. Fractions were collected from the top of the gradient and analyzed for the presence of Vam3p, Vph1p, and the endosomal t-SNARE, Pep12p. In both gradients, Vph1p fractionated primarily in the low density fractions (1–4) of the gradient, as is typical for vacuolar membrane proteins (Darsow et al., 1997) (Fig. 4 A). In contrast, Pep12p fractionated exclusively in more dense regions of the gradient (fractions 5–7), indicating that distinct separation of vacuoles and endosomes was achieved in these gradients (Fig. 4 B). As expected, wild-type Vam3p primarily co-fractionated with Vph1p in the first four fractions of the gradient. However, while a small percentage of Vam3pL160P also fractionated in the top (vacuolar) region of the gradient, the majority of the mutant Vam3 protein (∼80%) was recovered in more dense fractions (5–7), which also contained Pep12p (Fig. 4 B). Thus, while some Vam3pL160P is localized to the vacuole, most of the protein appears to reside in a non-vacuolar compartment that co-fractionates with Pep12p-containing endosomes.


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.  Cleared cell lysates generated  from vam3Δ (TDY2) cells  containing either wild-type  VAM3 (pVAM3.414) or the  vam3L160P mutant (pVAM3L160P.414) were loaded onto  the top of an Accudenz step  gradient and centrifuged to  equilibrium. Fractions 1–12  were collected from the top  of the gradient. Proteins  were precipitated from the  fractions and then separated  by SDS-PAGE and transferred to nitrocellulose.  Vam3p, Vph1p, and Pep12p  were detected by immunoblotting and visualized by  ECL fluorography. The distribution of both wild-type and  mutant (Vam3pL160P) Vam3p  and Vph1p are shown graphically in A and the colocalization of Vam3pL160P and  Pep12p are shown in B.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Localization of Vam3 mutant proteins. Cleared cell lysates generated from vam3Δ (TDY2) cells containing either wild-type VAM3 (pVAM3.414) or the vam3L160P mutant (pVAM3L160P.414) were loaded onto the top of an Accudenz step gradient and centrifuged to equilibrium. Fractions 1–12 were collected from the top of the gradient. Proteins were precipitated from the fractions and then separated by SDS-PAGE and transferred to nitrocellulose. Vam3p, Vph1p, and Pep12p were detected by immunoblotting and visualized by ECL fluorography. The distribution of both wild-type and mutant (Vam3pL160P) Vam3p and Vph1p are shown graphically in A and the colocalization of Vam3pL160P and Pep12p are shown in B.
Mentions: The ability of the vam3 mutants to suppress pep12Δ vacuolar protein sorting defects suggests that mutant Vam3 proteins are at least partially localized to the pre-vacuolar endosome, the site of Pep12p function. To examine the localization of the Vam3 mutant proteins, we fractionated cells and examined the distribution of the Vam3 mutant proteins relative to wild-type Vam3p by differential centrifugation. Vam3p fractionates exclusively in a low speed (P13) pellet fraction by differential centrifugation, which is consistent with its vacuolar localization (Darsow et al., 1997). However, the mutant forms of Vam3p fractionated differently than the wild-type protein, with a significant portion of the proteins localized to a high speed P100 pellet, (data not shown) which indicated that at least a portion of the mutant proteins localized to a non-vacuolar fraction. Although the P13 fraction is enriched in vacuoles, other compartments also fractionate in the P13. For example, the endosomal t-SNARE Pep12p typically exhibits a 40% P13/60% P100 fractionation pattern, even though this protein is not localized to the vacuole (Becherer et al., 1996). To further resolve vacuoles away from other P13 material, vam3Δ cells harboring either single-copy wild-type VAM3 or the vam3L160P mutant were analyzed by equilibrium density gradient fractionation. Cleared spheroplast lysates were applied to the top of Accudenz step gradients, which were then centrifuged to equilibrium. Fractions were collected from the top of the gradient and analyzed for the presence of Vam3p, Vph1p, and the endosomal t-SNARE, Pep12p. In both gradients, Vph1p fractionated primarily in the low density fractions (1–4) of the gradient, as is typical for vacuolar membrane proteins (Darsow et al., 1997) (Fig. 4 A). In contrast, Pep12p fractionated exclusively in more dense regions of the gradient (fractions 5–7), indicating that distinct separation of vacuoles and endosomes was achieved in these gradients (Fig. 4 B). As expected, wild-type Vam3p primarily co-fractionated with Vph1p in the first four fractions of the gradient. However, while a small percentage of Vam3pL160P also fractionated in the top (vacuolar) region of the gradient, the majority of the mutant Vam3 protein (∼80%) was recovered in more dense fractions (5–7), which also contained Pep12p (Fig. 4 B). Thus, while some Vam3pL160P is localized to the vacuole, most of the protein appears to reside in a non-vacuolar compartment that co-fractionates with Pep12p-containing endosomes.

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