The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains

Abstract

The product of the VPS1 gene, Vpslp, is required for the sorting of soluble vacuolar proteins in the yeast Saccharomyces cerevisiae. We demonstrate here that Vpslp, which contains a consensus tripartite motif for guanine nucleotide binding, is capable of binding and hydrolyzing GTP. Vpslp is a member of a subfamily of large GTP-binding proteins whose members include the vertebrate Mx proteins, the yeast MGM1 protein, the Drosophila melanogaster shibire protein, and dynamin, a bovine brain protein that bundles microtubules in vitro. Disruption of microtubules did not affect the fidelity or kinetics of vacuolar protein sorting, indicating that Vpslp function is not dependent on microtubules. Based on mutational analyses, we propose a two-domain model for Vpslp function. When VPS1 was treated with hydroxylamine, half of all mutations isolated were found to be dominant negative with respect to vacuolar protein sorting. All of the dominant-negative mutations analyzed further mapped to the amino-terminal half of Vpslp and gave rise to full-length protein products. In contrast, recessive mutations gave rise to truncated or unstable protein products. Two large deletion mutations in VPS1 were created to further investigate Vpslp function. A mutant form of Vpslp lacking the carboxy-terminal half of the protein retained the capacity to bind GTP and did not interfere with sorting in a wild-type background. A mutant form of Vpslp lacking the entire GTP-binding domain interfered with vacuolar protein sorting in wild-type cells. We suggest that the amino-terminal domain of Vpslp provides a GTP-binding and hydrolyzing activity required for vacuolar protein sorting, and the carboxy-terminal domain mediates Vpslp association with an as yet unidentified component of the sorting apparatus.