SELECTIVE RETENTION OF SECRETORY PROTEINS IN THE YEAST ENDOPLASMIC-RETICULUM BY TREATMENT OF CELLS WITH A REDUCING AGENT

We have used four glycoproteins as markers to study how disulfide bond formation and protein folding effect the intracellular transport of p roteins in yeast. Under normal conditions, the vacuolar enzyme carboxy peptidase Y (CPY) and the secretory stress-protein hsp150 acquired dis ulfide bonds in the endoplasmic reticulum (ER). Treatment of living ce lls with the reducing agent dithiothreitol (DTT) prevented disulfide f ormation of newly synthesized CPY and hsp150, resulting in retention o f the proteins in the ER. When DTT was removed, the sulfhydryls were r eoxidized, and the transport of the proteins to their correct destinat ions was resumed. Even mature CPY, located in the vacuole, could be re duced with DTT, and reoxidized after removal of the drug. DTT treatmen t blocked intracellular transport of hsp150 only when present during t he synthesis and translocation of the protein. Reduction of folded hsp 150, accumulated in the ER due to a sec block prior to DTT treatment, did not inhibit its secretion. The Kar2p/BiP protein, a component of t he ER lumen, was found to be associated with fully translocated reduce d hsp150, but not with native hsp150, suggesting that Kar2p/BiP may be involved in the putative retention mechanism. The cysteine-free pro-a lpha-factor, and invertase which was shown to have free sulfhydryls, w ere secreted and modified similarly in the presence and absence of DTT , showing that the secretory pathway of yeast functioned under reducin g conditions.