Abstract
Most G-proteins require a guanine nucleotide exchange factor (GEF) to regulate a variety of critical cellular processes. Interestingly, a small number of G-proteins switch between the active and inactive forms without a GEF. Translation elongation factor 1A (eEF1A) normally requires the GEF eEF1Balpha to accelerate nucleotide dissociation. However, several mutant forms of eEF1A are functional independent of this essential regulator in vivo. GEF-independent eEF1A mutations localize close to the G-protein motifs that are crucial for nucleotide binding. 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 11-22-fold increase in rates of GDP dissociation from eEF1A compared with 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.
Highlights
GTPase activation factors stimulate GTP hydrolysis, resulting in the inactive GDP-bound form
Upon binding of the guanine nucleotide exchange factor (GEF) to the G-protein, the phosphate groups are released first, and the base of the entering nucleotide binds to the NKXD motif and displaces the GEF [4]
G-proteins could maintain rapid nucleotide release rates that lead to GEF independence in several ways
Summary
The binding affinity for mant-GDP or mant-GMMPNP to wild type or the mutant forms of eEF1A was measured by a fluorimetric titration assay using a FluoroMax-3 spectrofluorimeter (Horiba Jobin Yvon Inc.). Wild type or mutant forms of eEF1A (1 M) in 2.5 ml of binding buffer (10% glycerol, 50 mM Tris-HCl, pH 8.0, 50 mM KCl, and 5 mM MgCl2) were placed in a 10 ϫ 10 ϫ 40-mm quartz cuvette with a magnetic stirring bar. The protein concentrations of the purified fractions were determined from the measurement of their different spectra in 6 M guanidine HCl between pH 12.5 and pH 6.0. The stock solutions were diluted to 0.2– 0.4 mg/ml in purification buffer 1 (20 mM Tris, pH 7.5, 0.1 mM EDTA, pH 8.0, 25% glycerol, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and 1 g/ml aprotonin) to a total volume of 300 l in cells with a 0.1-cm path wavelength. All CD data were reported in degrees cm dmolϪ1 ([])
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