FIG. 5.

Characterization of ypt11^G40D and ypt11^1144N. (A) Two-hybrid interaction of mutant Ypt11p with Myo2p. (Top) Reporter cells (strain PJ69-4A) carrying HIS3 and ADE2 under the control of USA_GAL were transformed with pGBDU-C1-based constructs for USA_GAL-binding domain fusion of the indicated YPT11 or pGBDU-C1 for a control (GBD) and with pGAD-C1-based constructs for trans-activator domain fusion of MYO2 or pGAD-C1 for a control (GAD). Cells were streaked on SC medium lacking uracil, leucine, histidine, and adenine, where two-hybrid interactions were detected as cell growth. (Bottom) Relative amounts of the USA_GAL-binding domain (GBD) fusion of Ypt11p in the cells above (lane 1, control; lane 2, ypt11^G40D; lane 3, ypt11^1144N; lane 4, YPT11) were analyzed by Western analysis using anti-GBD antibodies, showing that the mutations in YPT11 did not affect the YPT11 expression from the constructs. (B) Effect of overexpression of mutant YPT11 on cells growth. (Top) ypt11Δ cells (strain yTO001) with a control plasmid (vector) or with plasmids for overexpression of indicated YPT11 under the control of the GAL1 promoter were spotted on SCGal plates lacking uracil and incubated at 30°C for 3 days. The cell suspension for the spot was diluted 10-fold from left to right. (Bottom) ypt11Δ cells (strain yTO001) with a control plasmid (lane 1) or with plasmids for overexpression of YPT11^G40D (lane 2), YPT11^1144N (lane 3), or wild-type YPT11 (lane 4) were cultured in SCGal medium lacking uracil for 2 h and harvested. The same amount of proteins in each cell lysate was analyzed by Western blotting using anti-GFP antibodies.