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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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Vacuolar protein  sorting in vam3  cells.  SEY6210 (WT), TDY1  (vam3Δ), and TDY1 cells  harboring single-copy complementing VAM3 plasmid  (pVAM3.414) were converted to spheroplasts, and  then pulse labeled with  [35S]cysteine/methionine for  10 min at 30°C. Chase medium containing nonradioactive cysteine and methionine was added and incubation was continued  for an additional 45 min. The spheroplasts were separated into  intracellular (I) and extracellular (E) fractions, and CPY was immunoprecipitated with specific polyclonal antibodies, resolved  by SDS-PAGE, and analyzed by autoradiography. The positions of  Golgi-modified precursor (p2) and mature (m) CPY are indicated.
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Figure 2: Vacuolar protein sorting in vam3 cells. SEY6210 (WT), TDY1 (vam3Δ), and TDY1 cells harboring single-copy complementing VAM3 plasmid (pVAM3.414) were converted to spheroplasts, and then pulse labeled with [35S]cysteine/methionine for 10 min at 30°C. Chase medium containing nonradioactive cysteine and methionine was added and incubation was continued for an additional 45 min. The spheroplasts were separated into intracellular (I) and extracellular (E) fractions, and CPY was immunoprecipitated with specific polyclonal antibodies, resolved by SDS-PAGE, and analyzed by autoradiography. The positions of Golgi-modified precursor (p2) and mature (m) CPY are indicated.

Mentions: The abnormal vacuolar morphology of vam3Δ cells suggested that both protein transport to the vacuole and vacuolar function may be compromised in these cells. Therefore, the ability of vam3Δ cells to properly transport newly synthesized resident vacuolar proteins was examined. Spheroplasts prepared from vam3Δ cells were pulse labeled for 10 min with [35S]cysteine/methionine to label newly synthesized proteins, and then were chased with unlabeled cysteine/methionine for 45 min. The spheroplasts were separated into intracellular and extracellular fractions. CPY was immunoprecipitated from the samples and analyzed by SDS-PAGE and autoradiography. The biosynthesis of vacuolar proteins such as CPY can be monitored by posttranslational modifications that correlate with transport through the secretory and vacuolar protein sorting pathways. In the case of CPY, p1CPY is generated as a result of core glycosyl modifications in the ER, which are then elongated in the Golgi complex to generate the slightly larger form, p2CPY. Upon delivery to the vacuole, the precursor CPY is cleaved to generate the mature active form of the enzyme, mCPY. As shown in Fig. 2, wild-type cells properly delivered CPY to the vacuole as shown by the presence of mature CPY in the intracellular fraction (Fig. 2, lanes 1 and 2). In vam3Δ cells, CPY accumulated as the Golgi-modified p2 form, ∼50% of which was secreted into the extracellular fraction (Fig. 2, lanes 3 and 4). A small portion (∼10%) of the CPY that remained in the intracellular fraction was seen as an apparently misprocessed form that migrated as a species slightly smaller than p2CPY, which eventually was processed to mature CPY after prolonged chase (t1/2 > 60 min). The introduction of VAM3 on a single copy, centromere (CEN)–based plasmid complemented the CPY maturation defect of the vam3Δ mutant cells (Fig. 2, lanes 5 and 6).


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)

Vacuolar protein  sorting in vam3  cells.  SEY6210 (WT), TDY1  (vam3Δ), and TDY1 cells  harboring single-copy complementing VAM3 plasmid  (pVAM3.414) were converted to spheroplasts, and  then pulse labeled with  [35S]cysteine/methionine for  10 min at 30°C. Chase medium containing nonradioactive cysteine and methionine was added and incubation was continued  for an additional 45 min. The spheroplasts were separated into  intracellular (I) and extracellular (E) fractions, and CPY was immunoprecipitated with specific polyclonal antibodies, resolved  by SDS-PAGE, and analyzed by autoradiography. The positions of  Golgi-modified precursor (p2) and mature (m) CPY are indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2141632&req=5

Figure 2: Vacuolar protein sorting in vam3 cells. SEY6210 (WT), TDY1 (vam3Δ), and TDY1 cells harboring single-copy complementing VAM3 plasmid (pVAM3.414) were converted to spheroplasts, and then pulse labeled with [35S]cysteine/methionine for 10 min at 30°C. Chase medium containing nonradioactive cysteine and methionine was added and incubation was continued for an additional 45 min. The spheroplasts were separated into intracellular (I) and extracellular (E) fractions, and CPY was immunoprecipitated with specific polyclonal antibodies, resolved by SDS-PAGE, and analyzed by autoradiography. The positions of Golgi-modified precursor (p2) and mature (m) CPY are indicated.
Mentions: The abnormal vacuolar morphology of vam3Δ cells suggested that both protein transport to the vacuole and vacuolar function may be compromised in these cells. Therefore, the ability of vam3Δ cells to properly transport newly synthesized resident vacuolar proteins was examined. Spheroplasts prepared from vam3Δ cells were pulse labeled for 10 min with [35S]cysteine/methionine to label newly synthesized proteins, and then were chased with unlabeled cysteine/methionine for 45 min. The spheroplasts were separated into intracellular and extracellular fractions. CPY was immunoprecipitated from the samples and analyzed by SDS-PAGE and autoradiography. The biosynthesis of vacuolar proteins such as CPY can be monitored by posttranslational modifications that correlate with transport through the secretory and vacuolar protein sorting pathways. In the case of CPY, p1CPY is generated as a result of core glycosyl modifications in the ER, which are then elongated in the Golgi complex to generate the slightly larger form, p2CPY. Upon delivery to the vacuole, the precursor CPY is cleaved to generate the mature active form of the enzyme, mCPY. As shown in Fig. 2, wild-type cells properly delivered CPY to the vacuole as shown by the presence of mature CPY in the intracellular fraction (Fig. 2, lanes 1 and 2). In vam3Δ cells, CPY accumulated as the Golgi-modified p2 form, ∼50% of which was secreted into the extracellular fraction (Fig. 2, lanes 3 and 4). A small portion (∼10%) of the CPY that remained in the intracellular fraction was seen as an apparently misprocessed form that migrated as a species slightly smaller than p2CPY, which eventually was processed to mature CPY after prolonged chase (t1/2 > 60 min). The introduction of VAM3 on a single copy, centromere (CEN)–based plasmid complemented the CPY maturation defect of the vam3Δ mutant cells (Fig. 2, lanes 5 and 6).

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