Figure 1.

Enzymological properties of the Arabidopsis heterotrimeric G-protein alpha subunit (AtGPA1) in comparison to human Gα_oA. (A) GTPγS binding rates of AtGPA1 and Gα_oA were measured using standard methods.⁶,²⁰ Data were fit using a single exponential function and rate constants are presented in min⁻¹. Note: the observed GTPγS binding rate (k_on) is equivalent to the rate of GDP release, as GDP release is the rate limiting step in this process.¹¹ (B) The GTP hydrolysis rates of AtGPA1 and Gα_oA were measured using single turnover GTP hydrolysis⁷⁸ (i.e., using Gα subunits preloaded with [γ-³²P]GTP). Data were fit using a single exponential function and rate constants are presented in min⁻¹. (C) Steady state GTP hydrolysis of 100 nM AtGPA1 was measured using [γ-³²P]GTP, as described.⁶ AtGPA1 was incubated with either buffer (dotted black line), 250 nM wild-type AtRGS1(black line), or 250 nM E320K AtRGS1(grey line). The latter protein serves as a negative control for this experiment as it harbors a glutamate-320 to lysine (E320K) charge-reversal mutation that cripples RGS domain-mediated GAP activity, as previously described.⁶,⁷⁹ A general phenomenon of RGS proteins is that steady state GTPase acceleration cannot be observed in the absence of a GEF⁸⁰ as GDP release (not GTP hydrolysis) is rate limiting step in the G-protein cycle.¹¹ However, in this case, the steady state GTPase rate of AtGPA1 is greatly accelerated in the presence of AtRGS1-mediated GAP activity, highlighting the fact that GDP release is not rate-limiting for this plant Gα subunit. Observed rate constants were calculated by linear regression: AtGPA1 532 cpm/min, + wild-type AtRGS1 9867 cpm/min, + E320K AtRGS1 579 cpm/min.