|About GSBS | FAQ | Job Opportunities | Search UMDNJ|
Sedide B. Ozturk
University of Nevada, Reno
Thesis Advisor: Terri Goss Kinzy, Ph.D.
Graduate Program in Molecular Genetics, Microbiology and Immunology
RWJMS Room 747
Wednesday, April 2, 2008
G-proteins play critical roles in many cellular processes and are regulated by accessory proteins that modulate the nucleotide bound state. Such proteins, including eukaryotic Translation Elongation Factor 1A (eEF1A), are frequently reactivated by guanine nucleotide exchange factors (GEFs). In the yeast Saccharomyces cerevisiae only the catalytic subunit of the GEF complex, eEF1BƒÑ, is essential for viability. The requirement for the TEF5 gene encoding eEF1BƒÑ can be suppressed by the presence of excess substrate, eEF1A. These cells, however, have defects in growth and translation. Two independent unbiased genetic screens performed to dissect the cause of these phenotypes yielded dominant suppressors that bypass the requirement for extra eEF1A. Surprisingly, all mutations are in the G-protein eEF1A and cluster in its GTP-binding domain. Five mutants were used to construct novel strains expressing only the eEF1A mutant at normal levels. These strains show no growth defects and little to no decrease in total translation, which raises questions as to the evolutionary expression of GEF complexity and other potential functions of this complex. Structural analysis of the mutations suggests that their mechanism of suppression depends on their unique effects on two major conserved elements of the G-proteins: the P-loop and NKXD nucleotide binding element. Kinetic analysis demonstrated that reduced GDP affinity correlates with wild type growth and high translation activities of GEF-independent mutants. Furthermore, the mutant forms show an 11 to 22-fold increase in rates of GDP dissociation from eEF1A compared to the wild type protein. All mutant forms have dramatically enhanced stability at elevated temperatures. This, coupled with data demonstrating that eEF1A is also more stable in the presence of nucleotides, suggests that both the GEF and nucleotide have stabilizing effects on eEF1A. The biochemical properties of these eEF1A mutants provide insight into the mechanism behind GEF-independent G-protein function.