FIG 6.

Ofd1 does not regulate Cph2N stability. (A) The N-terminal fragment of Cph2 is stabilized by farnesol. Cells of the WT strain carrying Mal2p-myc-CPH2 (HLY3968) from overnight cultures were inoculated 1:50 into YPM and grown at 30°C for 5 h. The log-phase cells were washed twice with water and inoculated into YPD at 30°C either with or without 40 μM farnesol. For hypoxia (0.2% O₂, 5% CO₂), cells from overnight culture were washed twice and inoculated 1:50 into YPM and grown for 5 h under 0.2% O₂ and 5% CO₂ at 30°C before glucose was added. Cells were collected at 30 and 60 min for Western blotting. (B). Ofd1C does not promote Cph2 degradation. WT cells carrying OFD1-1 and MAL2p-myc-CPH2 (HLY4225) were used to determine Cph2 stability under the same conditions as for panel A. (C) The ofd1 and ubr1 deletions do not block Cph2N degradation. WT, ofd1, or ubr1 cells containing Mal2p-myc-CPH2 (HHLY3968, HLY4208, and HLY4224) were used to determine Cph2 stability in air without farnesol as described for panel A.