Limits...
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.

Show MeSH

Related in: MedlinePlus

Vacuolar protein sorting in vam3tsf mutant cells. TDY1  (vam3Δ) cells transformed with either complementing plasmid  (pVAM3.414) or plasmid containing a temperature-sensitive for  function (tsf) allele of vam3 (pVAM3-6.414) were converted to  spheroplasts, and then incubated at either permissive (26°C) or  nonpermissive (38°C) temperature for 5 min. Cultures were labeled with [35S]cysteine/methionine for 10 min, and then chased  for an additional 45 min at the indicated temperature. The cultures were separated into intracellular (I) and extracellular (E)  fractions, and the vacuolar proteins CPY, PrA, CPS, and ALP  were immunoprecipitated from each fraction, resolved by SDS-PAGE, and followed by autoradiography. CPS samples were  treated with endoglycosidase H before electrophoresis. The positions of Golgi-modified precursor (p2, pro) and mature vacuolar  (m) proteins are indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2141632&req=5

Figure 3: Vacuolar protein sorting in vam3tsf mutant cells. TDY1 (vam3Δ) cells transformed with either complementing plasmid (pVAM3.414) or plasmid containing a temperature-sensitive for function (tsf) allele of vam3 (pVAM3-6.414) were converted to spheroplasts, and then incubated at either permissive (26°C) or nonpermissive (38°C) temperature for 5 min. Cultures were labeled with [35S]cysteine/methionine for 10 min, and then chased for an additional 45 min at the indicated temperature. The cultures were separated into intracellular (I) and extracellular (E) fractions, and the vacuolar proteins CPY, PrA, CPS, and ALP were immunoprecipitated from each fraction, resolved by SDS-PAGE, and followed by autoradiography. CPS samples were treated with endoglycosidase H before electrophoresis. The positions of Golgi-modified precursor (p2, pro) and mature vacuolar (m) proteins are indicated.

Mentions: If Vam3p is directly required for vacuolar protein transport, then inactivation of the protein (i.e., by temperature shift) would be expected to result in an immediate block in vacuolar protein processing. We tested vam3tsf cells for sorting of vacuolar hydrolases after a short incubation at nonpermissive temperature. Spheroplasts were prepared from both vam3tsf and wild-type cells. The cultures were split and half was incubated at 26°C while the other half was shifted to 38°C for 5 min. Each culture was then pulse labeled for 10 min with [35S]cysteine/methionine and chased with unlabeled cysteine/methionine for 45 min. Samples were harvested and separated into intracellular and extracellular fractions. Vacuolar proteins were immunoprecipitated with specific antibodies and analyzed by SDS-PAGE. As shown in Fig. 3, at the permissive temperature of 26°C, vam3tsf cells matured CPY in a manner indistinguishable from wild-type cells (Fig. 3, lanes 1–4). However, at 38°C, a rapid block in the maturation of CPY was observed (Fig. 3, lanes 5 and 6). Approximately 60% of CPY accumulated as the Golgi-modified p2 precursor form. The remaining 40% of CPY was processed aberrantly and accumulated intracellularly. Less than 5% of the newly synthesized CPY was secreted to the extracellular media fraction. Similar results were observed for another soluble vacuolar hydrolase, PrA. We also examined the processing of two vacuolar membrane proteins, ALP and CPS. In vam3tsf cells at permissive temperature, the processing of both ALP and CPS occurred in a manner identical to wild-type cells (Fig. 3, lanes 1–4). However, in vam3tsf cells after a 5-min incubation at nonpermissive temperature, both proteins were completely blocked as the Golgi-modified precursor forms (Fig. 3, lanes 5 and 6). Vam3p thus appears to play an essential role in vacuolar protein transport of both soluble and integral membrane proteins.


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 vam3tsf mutant cells. TDY1  (vam3Δ) cells transformed with either complementing plasmid  (pVAM3.414) or plasmid containing a temperature-sensitive for  function (tsf) allele of vam3 (pVAM3-6.414) were converted to  spheroplasts, and then incubated at either permissive (26°C) or  nonpermissive (38°C) temperature for 5 min. Cultures were labeled with [35S]cysteine/methionine for 10 min, and then chased  for an additional 45 min at the indicated temperature. The cultures were separated into intracellular (I) and extracellular (E)  fractions, and the vacuolar proteins CPY, PrA, CPS, and ALP  were immunoprecipitated from each fraction, resolved by SDS-PAGE, and followed by autoradiography. CPS samples were  treated with endoglycosidase H before electrophoresis. The positions of Golgi-modified precursor (p2, pro) and mature vacuolar  (m) proteins are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Vacuolar protein sorting in vam3tsf mutant cells. TDY1 (vam3Δ) cells transformed with either complementing plasmid (pVAM3.414) or plasmid containing a temperature-sensitive for function (tsf) allele of vam3 (pVAM3-6.414) were converted to spheroplasts, and then incubated at either permissive (26°C) or nonpermissive (38°C) temperature for 5 min. Cultures were labeled with [35S]cysteine/methionine for 10 min, and then chased for an additional 45 min at the indicated temperature. The cultures were separated into intracellular (I) and extracellular (E) fractions, and the vacuolar proteins CPY, PrA, CPS, and ALP were immunoprecipitated from each fraction, resolved by SDS-PAGE, and followed by autoradiography. CPS samples were treated with endoglycosidase H before electrophoresis. The positions of Golgi-modified precursor (p2, pro) and mature vacuolar (m) proteins are indicated.
Mentions: If Vam3p is directly required for vacuolar protein transport, then inactivation of the protein (i.e., by temperature shift) would be expected to result in an immediate block in vacuolar protein processing. We tested vam3tsf cells for sorting of vacuolar hydrolases after a short incubation at nonpermissive temperature. Spheroplasts were prepared from both vam3tsf and wild-type cells. The cultures were split and half was incubated at 26°C while the other half was shifted to 38°C for 5 min. Each culture was then pulse labeled for 10 min with [35S]cysteine/methionine and chased with unlabeled cysteine/methionine for 45 min. Samples were harvested and separated into intracellular and extracellular fractions. Vacuolar proteins were immunoprecipitated with specific antibodies and analyzed by SDS-PAGE. As shown in Fig. 3, at the permissive temperature of 26°C, vam3tsf cells matured CPY in a manner indistinguishable from wild-type cells (Fig. 3, lanes 1–4). However, at 38°C, a rapid block in the maturation of CPY was observed (Fig. 3, lanes 5 and 6). Approximately 60% of CPY accumulated as the Golgi-modified p2 precursor form. The remaining 40% of CPY was processed aberrantly and accumulated intracellularly. Less than 5% of the newly synthesized CPY was secreted to the extracellular media fraction. Similar results were observed for another soluble vacuolar hydrolase, PrA. We also examined the processing of two vacuolar membrane proteins, ALP and CPS. In vam3tsf cells at permissive temperature, the processing of both ALP and CPS occurred in a manner identical to wild-type cells (Fig. 3, lanes 1–4). However, in vam3tsf cells after a 5-min incubation at nonpermissive temperature, both proteins were completely blocked as the Golgi-modified precursor forms (Fig. 3, lanes 5 and 6). Vam3p thus appears to play an essential role in vacuolar protein transport of both soluble and integral membrane proteins.

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