FIGURE 5.
Cdc50p contributes directly and specifically to Drs2p phosphoenzyme formation. A, wild type (CDC50) and Δcdc50 mutant strains co-transfected with pH2-DRS2 and pCDC50myc, pCRF1myc, pLEM3myc, pLEM3, or empty vector were analyzed by immunoblotting as indicated. B, quantification of H2-Drs2p levels in transfected wild type and Δcdc50 mutant strains under A. Data presented are means ± S.D. (error bars) of four independent experiments. EV, empty vector. C, H2-Drs2p was co-expressed with either Lem3p-Myc or Cdc50p-Myc in a Δcdc50 mutant strain and then subjected to tandem affinity purification as in Fig. 2A. Following elution from α-HA-coupled beads (EHA), the purified complexes were bound to Ni2+-NTA beads. Unbound (FTNTA) and NTA-bound (BNTA) fractions were collected and analyzed by immunoblotting using α-HA and α-Myc antibodies. D, EHA eluates were subjected to α-Myc immunoaffinity chromatography as in Fig. 4A to purify 100% Cdc50 subunit-bound Drs2p. EMYC, eluate fraction. E, equal amounts of 100% Cdc50 subunit-bound Drs2p were labeled with 2 μm [γ-32P]ATP as in Fig. 3A. F, quantification of VO4-sensitive phosphoenzyme formation in the Drs2p-Cdc50p and Drs2p-Lem3p complexes. Data presented are means ± S.D. (error bars) of three independent experiments.