FIGURE 4.

Re-directing Psd1p to the ER in vivo. A, schematic of in vivo Psd1 and CPY*mPsd1 constructs. MTS, mitochondrial targeting sequence; TM, transmembrane domain; CPY, ER targeting signal of carboxypeptidase Y; NXS, inserted N-glycosylation signal. B, subcellular fractions were prepared from psd1Δpsd2Δ yeast (−) and psd1Δpsd2Δ yeast transformed with Psd1 or CPY*mPsd1 grown in YPD via a series of differential centrifugations. Thirty micrograms of each fraction were resolved by SDS-PAGE and immunoblotted for both subunits of Psd1p (α and β) and the following organelle markers; Qcr6p (mitochondria), Sec62p (ER), and Hsp70p (cytosol). C, whole cell extracts derived from psd1Δpsd2Δ yeast transformed with Psd1 or CPY*mPsd1 grown in YPD were mock-treated (M) or treated (+) with EndoH for 4 h at 37 °C. The β and α subunits of Psd1p were detected by immunoblot with Aac2p serving as a loading control. For CPY*mPsd1, glycosylated (+CHO) and deglycosylated (−CHO) β subunits are indicated.