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A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole.

Darsow T, Rieder SE, Emr SD - J. Cell Biol. (1997)

Bottom Line: Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation.Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Delta mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Delta mutant phenotypes.This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.

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.

ABSTRACT
Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283-amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3(tsf)). Upon shifting the vam3(tsf) mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3(tsf) cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift, vam3(tsf) cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Delta mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Delta mutant phenotypes. This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.

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Analysis of API maturation in vam3tsf cells. Wild-type  (SEY6210) cells and TDY1 (vam3Δ) cells carrying a vam3tsf plasmid (pVAM3-6.414) were incubated at 38°C for 5 min, labeled  with [35S]cysteine/methionine for 10 min, and then chased for the  indicated times. Equivalent volumes of labeled culture were harvested at the indicated time points of chase. API was recovered  from lysates by immunoprecipitation, subjected to SDS-PAGE,  and analyzed by autoradiography. The cytoplasmic precursor  (pr) and mature vacuolar (m) forms of API are indicated.
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Figure 4: Analysis of API maturation in vam3tsf cells. Wild-type (SEY6210) cells and TDY1 (vam3Δ) cells carrying a vam3tsf plasmid (pVAM3-6.414) were incubated at 38°C for 5 min, labeled with [35S]cysteine/methionine for 10 min, and then chased for the indicated times. Equivalent volumes of labeled culture were harvested at the indicated time points of chase. API was recovered from lysates by immunoprecipitation, subjected to SDS-PAGE, and analyzed by autoradiography. The cytoplasmic precursor (pr) and mature vacuolar (m) forms of API are indicated.

Mentions: Some newly synthesized vacuolar proteins do not reach the vacuole via the secretory pathway but instead follow a direct cytosol to vacuole delivery pathway (Klionsky et al., 1992). The precursor form of API is synthesized in the cytoplasm and transported directly into the vacuole where the amino-terminal precursor is cleaved, producing the mature form of the enzyme (Klionsky et al., 1992). Many mutants defective for the processing of API (cvt mutants) are also defective in autophagy (aut and apg mutants), indicating that delivery of API to the vacuole may be mediated by an autophagic mechanism (i.e., macroautophagy) (Harding et al., 1996; Scott et al., 1996). The processing of API was examined in vam3tsf cells to determine if Vam3p is required for delivery of API to the vacuole. Wild-type and vam3tsf cells were shifted to nonpermissive temperature for 5 min and then labeled for 10 min. Samples were harvested after 0, 30, 60, 90, and 120 min of chase. The maturation of API was analyzed by immunoprecipitation, SDS-PAGE, and autoradiography. As shown in Fig. 4, cells containing the wild-type copy of VAM3 matured API with normal kinetics; essentially all API was processed to the mature vacuolar form after 120 min of chase. However, vam3tsf cells at nonpermissive temperature (38°C) accumulated newly synthesized API in its precursor form, with virtually no maturation of the protein, even after 120 min of chase. In vam3tsf cells at the permissive temperature of 26°C, API was also matured with kinetics comparable to that of wild-type cells (data not shown). The defect in API processing observed in vam3tsf cells at nonpermissive temperature indicates that Vam3p is directly required for the delivery of API to the vacuole.


A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole.

Darsow T, Rieder SE, Emr SD - J. Cell Biol. (1997)

Analysis of API maturation in vam3tsf cells. Wild-type  (SEY6210) cells and TDY1 (vam3Δ) cells carrying a vam3tsf plasmid (pVAM3-6.414) were incubated at 38°C for 5 min, labeled  with [35S]cysteine/methionine for 10 min, and then chased for the  indicated times. Equivalent volumes of labeled culture were harvested at the indicated time points of chase. API was recovered  from lysates by immunoprecipitation, subjected to SDS-PAGE,  and analyzed by autoradiography. The cytoplasmic precursor  (pr) and mature vacuolar (m) forms of API are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141632&req=5

Figure 4: Analysis of API maturation in vam3tsf cells. Wild-type (SEY6210) cells and TDY1 (vam3Δ) cells carrying a vam3tsf plasmid (pVAM3-6.414) were incubated at 38°C for 5 min, labeled with [35S]cysteine/methionine for 10 min, and then chased for the indicated times. Equivalent volumes of labeled culture were harvested at the indicated time points of chase. API was recovered from lysates by immunoprecipitation, subjected to SDS-PAGE, and analyzed by autoradiography. The cytoplasmic precursor (pr) and mature vacuolar (m) forms of API are indicated.
Mentions: Some newly synthesized vacuolar proteins do not reach the vacuole via the secretory pathway but instead follow a direct cytosol to vacuole delivery pathway (Klionsky et al., 1992). The precursor form of API is synthesized in the cytoplasm and transported directly into the vacuole where the amino-terminal precursor is cleaved, producing the mature form of the enzyme (Klionsky et al., 1992). Many mutants defective for the processing of API (cvt mutants) are also defective in autophagy (aut and apg mutants), indicating that delivery of API to the vacuole may be mediated by an autophagic mechanism (i.e., macroautophagy) (Harding et al., 1996; Scott et al., 1996). The processing of API was examined in vam3tsf cells to determine if Vam3p is required for delivery of API to the vacuole. Wild-type and vam3tsf cells were shifted to nonpermissive temperature for 5 min and then labeled for 10 min. Samples were harvested after 0, 30, 60, 90, and 120 min of chase. The maturation of API was analyzed by immunoprecipitation, SDS-PAGE, and autoradiography. As shown in Fig. 4, cells containing the wild-type copy of VAM3 matured API with normal kinetics; essentially all API was processed to the mature vacuolar form after 120 min of chase. However, vam3tsf cells at nonpermissive temperature (38°C) accumulated newly synthesized API in its precursor form, with virtually no maturation of the protein, even after 120 min of chase. In vam3tsf cells at the permissive temperature of 26°C, API was also matured with kinetics comparable to that of wild-type cells (data not shown). The defect in API processing observed in vam3tsf cells at nonpermissive temperature indicates that Vam3p is directly required for the delivery of API to the vacuole.

Bottom Line: Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation.Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Delta mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Delta mutant phenotypes.This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.

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.

ABSTRACT
Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283-amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3(tsf)). Upon shifting the vam3(tsf) mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3(tsf) cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift, vam3(tsf) cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Delta mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Delta mutant phenotypes. This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.

Show MeSH
Related in: MedlinePlus