Yeast exocytic v-SNAREs confer endocytosis.
In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1 (SNC1(ala43)), are deficient in the endocytic uptake of components from the cell surface. We found that both SNC and temperature-shifted SNC1(ala43) yeast are deficient in their ability to deliver the soluble dye FM4-64 to the vacuole. Under conditions in which vesicles accumulate, FM4-64 stained primarily the cytoplasm as well as fragmented vacuoles. In addition, alpha-factor-stimulated endocytosis of the alpha-factor receptor, Ste2, was fully blocked, as evidenced using a Ste2-green fluorescent protein fusion protein as well as metabolic labeling studies. This suggests a direct role for Snc v-SNAREs in the retrieval of membrane proteins from the cell surface. Moreover, this idea is supported by genetic and physical data that demonstrate functional interactions with t-SNAREs that confer endosomal transport (e.g., Tlg1,2). Notably, Snc1(ala43) was found to be nonfunctional in cells lacking Tlg1 or Tlg2. Thus, we propose that synaptobrevin/VAMP family members are engaged in anterograde and retrograde protein sorting steps between the Golgi and the plasma membrane.
Gurunathan
S
S
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
Chapman-Shimshoni
D
D
Trajkovic
S
S
Gerst
J E
JE
D001483:Base Sequence; D003001:Cloning, Molecular; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology*; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology*; D005838:Genotype; D000072235:Mating Factor; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000502:physiology; D050683:R-SNARE Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014617:Vacuoles / Q000502:physiology*
D005656:Fungal Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D050683:R-SNARE Proteins; D011994:Recombinant Proteins; C083241:SNC1 protein, S cerevisiae; C083242:SNC2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor