Figure 6.

LRG1 is a RHO1–specific GTPase activating protein. (A) Overexpression of wild type as well as dominant-active (rho-1^G15V) and dominant-negative (rho-1^E41I) rho-1 alleles, but not of any of the other Rho proteins was sufficient to overcome the morphological defect of lrg-1(12-20) at restrictive temperature. Bar, 50 μm. (B) Death phenotypes of wild-type cells transformed with the overexpression constructs of the indicated RHO1 alleles. (C) Conidia (left side) of a heterocaryotic Δrho-1::hph^R + rho-1⁺ strain were germinated on minimal media supplemented with hygromycin. Spores harboring only the deletion nucleus germinated isotropically (red arrows), whereas heterokaryotic cells were able to produce polar germ tubes (blue arrowheads). Bar, 20 μm. Ascospore progeny (right side) of Δrho-1::hph^R + rho-1⁺× wild-type crosses predominatly germinated apolarly on medium supplemented with hygromycin (top image). Bar, 20 μm. Rarely (<1 of 1000 ascospores) we observed growing hyg^R colonies, which then displayed a high rate of cell lysis (bottom image). Bar, 200 μm. (D) Intrinsic and LRG1^650–1035 stimulated GTPase activities of the six Rho proteins. Bacterially expressed and affinity-purified Rho proteins were preloaded with [γ-32-P]GTP, and the reactions started by the addition of LRG1^650–1035 or buffer. GTPase activities were determined by measuring remaining RHO-bound [γ-³²P]GTP after 8 min at 25°C (see Materials and Methods). (E) Kinetics of in vitro RHO1 GTPase activity in the presence and absence on LRG1^650–1035.