Yeast Exocytic v-SNAREs Confer Endocytosis

Abstract

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, ␣-factor-stimulated endocytosis of the ␣-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.

t-SNARE Phosphorylation Regulates Endocytosis in Yeast

“…To create snc tlg vbm1 yeast, the snc vbm1 strains DD1 and MM1 were cured of their GAL-SNC1-containing plasmids and transformed with linearized DNA fragments isolated from the enzymatic digestion of pTLG1L and pTLG2T (Gurunathan et al, 2000). Transformants were selected and the disruptions verified by PCR analysis.…”

“…The Snc exocytic v-SNAREs also mediate endocytosis (Gurunathan et al, 2000). In snc null cells or cells possessing a temperature-sensitive allele of SNC1 (snc1 ala43 ) that were shifted to restrictive temperatures, uptake of the soluble dye FM4-64 and the Ste2 mating factor receptor to the vacuole is abolished.…”

“…Appropriately, their endocytic functions are likely to be dependent on two different t-SNAREs, Tlg1 and Tlg2, which are trans-Golgi and endosomal t-SNAREs involved in endosomal protein sorting (Abeliovich et al, 1998;Holthuis et al, 1998a;Seron et al, 1998;Coe et al, 1999;Abeliovich et al, 1999). This is because Snc proteins coimmunoprecipitate with the Tlg t-SNAREs (Abeliovich et al, 1998;Holthuis et al, 1998a); Snc1 ala43 is nonfunctional in their absence (Gurunathan et al, 2000); and snc tlg1 and snc tlg2 strains show synthetically enhanced phenotypes (Gurunathan et al, 2000). Moreover, Tlg1 and 2 have been recently shown to bind Snc v-SNAREs and to mediate an in vitro fusion event similar to endocytosis (Paumet et al, 2001).…”

Sphingolipids and membrane biology as determined from genetic models

“…In yeast, this process is mediated in part by membrane-associated proteins called SNAREs, which are encoded by the SNC genes [65][66][67]. The disruption of genes encoding the SNARE proteins can be detrimental to the organism or even lethal [68,69].…”

“…The SNC1 and SNC2 genes encode snc1p and snc2p respectively, which are archetypal v-SNARES involved in the exocytosis process [68,69,71,74]. The mutants cease growth at 38°C, and at 37°C they accumulate secretory vesicles and show defective protein secretion.…”