Mutational analysis of the yeast nucleotide exchange factor Mge1p

Mge1p is an essential nucleotide exchange factor, expressed by the budding yeast Saccharomyces cerevisiae, that catalyzes the release of ADP from Hsp70 molecules. Without such catalysis the Hsp70 reaction cycle comes to a halt. There is intriguing evidence that Mge1p and its homologues are more than just nucleotide exchange factors; specifically, these proteins may also be molecular thermosensors. In this study, a structure-function analysis of Mge1p was conducted to ascertain the functional significance of the putative thermosensing domain of Mge1p, which comprises the firstsimilar to66 N-terminal residues of the protein. We show that (i) yeast cells that express Mge1pDelta66 exhibit a severe growth defect compared to cells expressing wt Mge1p; and (ii) preliminary results from agenetic screen reveal that mutating Ile102 to Asn in the Mge1pDelta66 protein partially restores cell growth. To further elucidate the role of the 66 N-terminal residues, in the future we plan to isolate Mge1pDelta66 and variants that contain suppressor mutations and conduct biophysical studies that probe the binding of these proteins to mtHsp70.