Figure 7.

LRG1 is affecting several RHO1-dependent effector pathways. (A) The indicated strains were grown at permissive temperature and shifted to 37°C for 10 h (top) or germinated at restrictive temperature for 15 h (bottom) to illustrate the synthetic nature of the two mutations. Bar, 40 μm. (B) lrg-1(12-20), gs-1(8-6), and lrg-1(12-20);gs-1(8-6) grown at permissive temperature are hyposensitive against the β1,3-glucan synthase inhibitor caspofungin. (C) Growth of the indicated strains for 48 h at 32°C on plates supplemented with the kinase inhibitors staurosporine, KT5720, or cercosporamide. The growth defects of lrg-1(12-20) were suppressed by staurosporine and cercosporamide, but not by KT5720. The growth of wild-type cells was not affected by the mentioned conditions. One example of three consistent experiments is shown. (D) Total soluble protein was extracted from wild type and lrg-1(12-20) shifted to 37°C for the indicated times. The blot was probed with anti-phospho-ERK (α-P-ERK) antibody to detect activated MAK1 (top panel) induced by the temperature stress. A replicate was probed with anti-ERK to confirm equal loading (bottom panel). Wild type but not lrg-1(12-20) showed stress-induced MAK1 activation after 20 min at 37°C. (E) MAK1 phosphorylation induced by the addition of 7 mM H₂O₂ for 30 min to wild type and lrg-1(12-20) indicate that the response capacity of the MAK1 pathway is not affected in lrg-1 mutants. One example of five consistent experiments is shown in D and E (originating from individually treated biological cultures and independent protein preparations). (F) lrg-1(12-20) grown at permissive conditions is hypersensitive against the actin-depolymerizing drug latrunculin A.