Figure 9.

Bul1p and Bul2p regulate Gap1p activity in conjunction with Rsp5p. (A) Wild-type (CKY4), rsp5-1 (CKY712), lst4Δ (CKY695), rsp5-1 lst4Δ (CKY713), bul1Δ bul2Δ (CKY698), and rsp5-1 bul1Δ bul2Δ (CKY714) strains were cultured in minimal medium (SD Ammonia) at 34°C. (B) Wild-type (CKY4) and rsp5-1 (CKY712) strains were transformed with a multicopy plasmin carrying BUL1 (pCK228) or the corresponding vector. Transformants were grown at 34°C in minimal medium (SD Ammonia). (D) Wild-type (CKY4), bul1Δ bul2Δ (CKY698), and lst4Δ bul1Δ bul2Δ (CKY699) strains were transformed with a low copy vector carrying BUL1 (pCK249), bul1^{P157Q, P158A} (pHY37), or the corresponding vector. Transformants were cultured in minimal medium (SD Ammonia) at 24°C. (A, B, and D) Strains were assayed for [¹⁴C]citrulline uptake and values are expressed as a percentage of the relevant wild-type strain. The data represent the mean for three independent experiments (A), or three separate transformants (B and D); error bars represent one standard deviation. (C) Wild-type (CKY4), rsp5-1 (CKY712), bul1Δ bul2Δ (CKY698), and rsp5-1 bul1Δ bul2Δ (CKY714) strains were transformed with pHA-GAP1 (pPL257), and protein extracts made from exponentially growing cultures in minimal medium (SD Ammonia) at 24°C or 34°C were subject to SDS-PAGE and Western blotting with anti-HA antibody. 12.5 times more material was loaded onto this gel compared with the gel in Fig. 3, in order to visualize the ubiquitinated forms of HA-Gap1p, which are denoted with an asterisk.