FIGURE 5.

Novel mutant of Sro7, sro7-R189D,R222D fails to induce clustering and cell lethality in vivo. A, schematic and surface-filling model show the four new, surface-exposed, conserved charge-reversal Sro7 mutants as follows: red, Sro7-R189D,R222D; yellow, Sro7-K196E,R210D; light blue, Sro7-K794E,H797E,K798E; violet, Sro7-K576E. B, wild type and sec15-1 mutant strains were transformed with plasmids, (CEN) overexpressing Sro7, or the charge reversal mutants from a GAL promoter. Three individual colonies were picked and transferred to selective media in the presence of glucose or galactose. Equal absorbance units of the strains induced in galactose were harvested, washed in Tris (10 mm) and azide (20 mm), and lysed by glass bead lysis before subjecting to Western blot analysis. C, wild type cells containing plasmids expressing SRO7 or sro7-D189,D222 from a GAL-inducible promoter (CEN) or vector only were grown in selective media and induced for 6 h in galactose before fixing and processing for immunofluorescence analysis using monoclonal Sec4 and polyclonal Sro7 antibodies. Scale bar, 2 μm.