Yeast Ysl2p, Homologous to Sec7 Domain Guanine Nucleotide Exchange Factors, Functions in Endocytosis and Maintenance of Vacuole Integrity and Interacts with the Arf-Like Small GTPase Arl1p

Abstract

We previously described the isolation of ysl2-1 due to its genetic interaction with Δypt51/vps21, a mutant with a deletion of the coding sequence for the yeast Rab5 homolog, which regulates endocytic traffic between early and late endosomes. Here we report that Ysl2p is a novel 186.8-kDa peripheral membrane protein homologous to members of the Sec7 family. We provide multiple genetic and biochemical evidence for an interaction between Ysl12p and the Arf-like protein Arl1p, consistent with a potential function as an Arf guanine nucleotide exchange factor (GEF). The temperature-sensitive alleles ysl2-307 and ysl2-316 are specifically defective in ligand-induced degradation of Ste2p and α-factor and exhibit vacuole fragmentation directly upon a shift to 37°C. In living cells, green fluorescent protein (GFP)-Ysl2p colocalizes with endocytic elements that accumulate FM4-64. The GFP-Ysl2p staining is sensitive to a mutation in VPS27 resulting in the formation of an aberrant class E compartment, but it is not affected by a sec7 mutation. Consistent with the idea that Ysl2p and Arl1p have closely related functions, Δarl1 cells are defective in endocytic transport and in vacuolar protein sorting.

We are very grateful to Cathy Jackson, Howard Riezman, Kathleen D'Hondt, Xin Nie, and Steve Elledge for providing plasmids and reagents used in this study. We acknowledge Sabine Frischmuth for assistance in the screen for temperature-sensitive mutants of ysl2, Evi Frei for help in the purification of ³⁵S-labeled α-factor, Yvonne Volkenstein for technical assistance, and Dieter H. Wolf for critical reading of the manuscript.

This work was supported by a grant from the DFG to B.S.-K. (Si 635/2-1).