Figure 4. Regulation of vesicular transport by diagnostic linear regions of ORP1S.

(A) Strain CMY136 transformed with pDB31 (pTPI-SUC2; encodes invertase under the control of the constitutive TPI1 promoter) and the indicated ORP1S fragment under the control of the GAL1 promoter were grown at 25 °C to mid-exponential phase in minimal medium with 2% (w/v) galactose as carbon source. Cells were washed, shifted to 37 °C for 2 h, and invertase secretion was measured as described in the Experimental section. The results are means±S.D. of three separate experiments performed in triplicate. Student's two-tailed t test was used to determine significant differences from the vector control (*P<0.05). (B) ORP1S constructs expressed from the GAL1 promoter were grown as in (A). Cells were pelleted by centrifugation and proteins contained in the supernatant were precipitated by adding trichloroacetic acid, collected by centrifugation, washed with 100% acetone, dried, and resuspended in SDS/PAGE sample buffer. Samples were resolved by SDS/PAGE and processed for CPY immunoblots. (C) Pelleted cells from (B) were processed for CPY immunoblots and compared with secreted CPY levels to compare the processing fate of internal compared with secreted CPY. Results in (B) and (C) are representative of three separate experiments.