Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation

Abstract

The role of the lumenal contents (reticuloplasm) of the endoplasmic reticulum (ER) in protein translocation was determined by in vitro analysis. Depletion of the reticuloplasm from mammalian rough microsomes revealed two distinct stages of the translocation reaction. The first stage, translocation up to and including signal peptide cleavage, was insensitive to the loss of the reticuloplasm, whereas the second stage, net transfer of the nascent chain into the ER lumen, was reticuloplasm dependent. In reticuloplasm-depleted membranes, signal-cleaved and glycosylated translocation intermediates were observed to transit free from the translocation channel to the cis, or cytoplasmic, side of the membrane. This translocation defect was complemented by reconstitution of lumenal proteins into depleted membranes. We propose that lumenal proteins are necessary for unidirectional protein translocation in mammalian ER.