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 proteins in yeast. Under normal conditions, the vacuolar enzyme carboxypeptidase Y (CPY) and the secretory stress-protein hsp150 acquired disulfide bonds in the endoplasmic reticulum (ER). Treatment of living cells with the reducing agent dithiothreitol (DTT) prevented disulfide formation of newly synthesized CPY and hsp150, resulting in retention of the proteins in the ER. When DTT was removed, the sulfhydryls were reoxidized, and the transport of the proteins to their correct destinations was resumed. Even mature CPY, located in the vacuole, could be reduced with DTT, and reoxidized after removal of the drug. DTT treatment blocked intracellular transport of hsp150 only when present during the synthesis and translocation of the protein. Reduction of folded hsp150, 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 the ER lumen, was found to be associated with fully translocated reduced hsp150, but not with native hsp150, suggesting that Kar2p/BiP may be involved in the putative retention mechanism. The cysteine-free pro-alpha-factor, and invertase which was shown to have free sulfhydryls, were secreted and modified similarly in the presence and absence of DTT, showing that the secretory pathway of yeast functioned under reducing conditions.