Nucleotide Binding to Yeast Arl1: Modulation by Mon2

Georgetown University, Washington DC

Investigators

Abstract

Monomeric G proteins control numerous events in cells by alternately binding GDP and GTP. Generally, G proteins are thought to be inactive when bound to GDP and active when bound to GTP. The Rosenwald laboratory has been studying one member of the G protein superfamily in detail in Saccharomyces cerevisiae, the Arl1 (Arf-like 1) protein, highly conserved over the course of eukaryotic evolution. Previous work has shown that Arl1 affects both membrane traffic and intracellular potassium levels. Current evidence suggests that Arl1, rather than acting as a two-stage switch, like canonical members of the superfamily, instead acts as a three-stage switch, cycling between a GDP-bound conformation (inactive), a nucleotide-free conformation (that affects ion homeostasis), and a GTP-bound conformation (that affects membrane traffic). Further, the Mon2 protein, also highly conserved over evolution, has been found to be an important regulator of nucleotide binding to Arl1, and thus of Arl1's function: the N-terminal end of the protein enhances nucleotide binding to Arl1, while the C-terminal end inhibits nucleotide binding. Mon2, the first enhancer of nucleotide binding discovered for any Arf-like protein in any species, thus represents a novel means for regulation of nucleotide binding to a G protein in vivo. In this project, the PI will investigate the mechanism by which Mon2 acts to control nucleotide binding to Arl1 both in vitro and in vivo. Current data are consistent with a model in which the N- and C-terminal regions of Mon2 bind to each other and to Arl1, first to stabilize nucleotide-free Arl1, then to load GTP onto Arl1. Talented undergraduate and graduate students of diverse backgrounds will specifically test this model, continuing the tradition established in the laboratory over the last several years, which has involved training of more than twenty undergraduates and four graduate students.