Figure 7.

Nucleotide binding properties and GTPase activity of Rab32. Bacterially purified GST fusion proteins were assayed for guanine nucleotide binding. (A) Schematic diagram depicting a mutation in the nucleotide binding domain of Rab32. A nucleotide binding site, GTPase domain, and sites of putative lipid modifications are indicated. (B) GST–Rab32 (2 μg) was preloaded with 2 μCi of [³H]GDP. GDP off rate is presented as percent GDP bound to Rab32 over time. (C) Nucleotide-depleted Rab32 was incubated with [γ³²P]GTP. The GTP on rate for GST–Rab32 was measured and is presented as the percentage of GTP bound over time. Amalgamated data from three experiments are presented. (D) Biochemical characterization of guanine nucleotide binding to recombinant wild-type GST–Rab32 and GST–Rab32T39N mutant. [³H]GDP binding (gray bars) and [γ³²P]GTP binding (black bars) to Rab32 and Rab32T39N are indicated. Results are presented as the percent nucleotide bound normalized to 1. 2 μg of GST recombinant proteins were used and shown in the inset. A representative experiment from three independent analyses is presented. (E) GST pulldowns of GDP-loaded Rab32 or GTPγS-loaded Rab32 in the presence of recombinant RII (1 μg). Proteins were separated by SDS-PAGE and transferred to nitrocellulose. (Top) Coprecipitation of RII was detected by Western blot using an anti-RII monoclonal antibody. (Bottom) Control experiments showing equal loading of proteins. (F) Yeast coexpressing pLexA Rab32 mutants and pACT2 RII 1–45 were assayed for growth on minimal media without histidine (left). As a toxicity control, yeast harboring these plasmids can grow in media supplemented with histidine (right).