Figure 1.

Full-length Sec2-GFP and truncated Sec2-59(1-374)-GFP exchange GDP for GTP on Sec4p with similar rates. Exchange reactions were performed to quantitate the Sec4p GTPγS on-rate. Anti-GFP immune complexes were generated from yeast expressing Sec2-GFP (NY2146), Sec2-59(1-374)-GFP (NY2147), or Sec2p (NY2145, control) and incubated with recombinant, partially purified, Sec4p and GTP/[³⁵S]GTPγS for various time points at 14°C. Reactions were stopped with ice cold exchange buffer and protein-associated radioactivity was quantitated on filters by scintillation counting. Exchange data were normalized to protein present in the immune complexes by subjecting immunoprecipitated material to Western analysis using the anti-GFP antibody. First order rate equations were used to calculate the enzymatic exchange rates. Sec2-GFP exchange rate was set at 100% (black circles). Sec2-59(1-374)-GFP (open circles) was found to exchange with 80% the activity of full-length Sec2-GFP (SEM = 6.9). Untagged Sec2p immune complexes were generated as a control (open squares). This reflects the intrinsic rate of exchange of Sec4p and was similar to protein A–Sepharose beads alone. Truncation of the COOH terminus of Sec2p does not dramatically affect its exchange activity and most likely does not account for the defect observed for Sec2-59(1-374)p expressing yeast.