Figure 3.

Expression of the mutant Glo3-R59K protein causes lethality. (A) Glo3-R59K-His6 was expressed in E. coli, purified using Ni²⁺/NTA-agarose, and used for an in vitro ArfGAP assay. The maximum activity observed corresponds to the hydrolysis of 82% of the total pool of radioactively labeled GTP-Arf per assay sample. Wild-type Glo3: (▪), Glo3-R59K: (•) (B) glo3Δ yeast cells harboring low-copy plasmid with the MET3pr-glo3-R59K gene were streaked for single colonies on medium containing methionine (Met+) to repress glo3-R59K expression or on medium lacking methionine (Met-) to induce glo3-R59K expression, and incubated at 30°C for 2 d. (C) Wild-type yeast cells harboring a low-copy plasmid with the MET3pr-glo3-R59K gene were streaked for single colonies on medium containing methionine (Met+) to repress glo3-R59K expression or on medium lacking methionine (Met-) to induce glo3-R59K expression, and incubated at 30°C for 2 days.