Figure 7.

Complementation of tpe2-1 by PDR3. The tpe2-1 strain LKY161 was transformed with either the plasmid pRS405 alone or the pRS405 plasmid containing the PDR3, pRS405-PDR3. Four transformants from each transformation were grown to mid-log phase in SDC medium lacking leucine at 23°C. (A) 1 ml of each culture was incubated at 30°C for 30 min, and M-C₆-NBD-PE internalization assays and fluorescence microscopy were performed as described in Materials and Methods. (a–c) tpe2-1 transformed with vector pRS405 alone. (d–f) tpe2-1 transformed with pRS405-PDR3. (a, b, d, and e) NBD fluorescence. (c and f) DIC optics. In a and d, a neutral density filter was used that attenuated the excitatory light by 95%. In b and e, the neutral density filter was removed to allow 100% of the excitatory light to reach the samples. The M-C₆-NBD-PE accumulation of the cells shown in this figure are representative of that observed for all four transformants analyzed. (B) 5 μl of each culture was spotted onto SDC plates lacking leucine and containing 0 (not shown), 0.25, 0.50, or 0.75 μg/ml cycloheximide and incubated at 30°C for 3 d.