Access of Proteinase K to Partially Translocated Nascent Polypeptides in Intact and Detergent-solubilized Membranes

We have used proteinase K as a probe to detect cytoplasmically and lumenally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were derived from preprolactin and the G protein of vesicular stomatitis virus and encoded nascent chains ranging between 64 and 200 amino acid residues long. Partially translocated nascent chains of 100 amino acid residues or less were insensitive to protease digestion from the external surface of the membrane while longer nascent chains were susceptible to digestion by externally added protease. We conclude that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, low molecular weight nascent chains were remarkably resistant to protease digestion even after detergent solubilization of the membrane. The protease resistant behavior of detergent solubilized nascent chains could be abolished by release of the polypeptide from the ribosome or by the addition of protein denaturants. We propose that the protease resistance of partially translocated nascent chains can be ascribed to components of the translocation apparatus that remain bound to the nascent chain after detergent solubilization of the membrane. T RANSPORT of nascent secretory proteins across the RER is mediated by multiple proteinaceous components which function in a defined sequence during each translocation event. In higher eukaryotes, the 54-kD subunit of the signal recognition particle (SRP) ~ binds to the amino-terminal signal sequence of the polypeptide upon emergence of the nascent chain from the large ribosomal subunit (17, 18, 37). In so doing, ribosomes synthesizing proteins destined for transport across the ER are selected for recognition by the SRP receptor (12) (or docking protein [20]). Displacement of SRP from the ribosome occurs upon interaction of the SRP-ribosome complex with the SRP receptor (11) in a reaction that is tightly coupled to the GTPdependent insertion of the signal sequence into the membrane (6). Events which mediate the subsequent transport of the polypeptide across the membrane are less well defined. Partially translocated nascent secretory polypeptides cannot be extracted from the microsomal membrane by high salt solutions or EDTA, but are extracted by protein denaturants (11), suggesting that both nascent chain attachment to the membrane and transport of the polypeptide across the bilayer may be mediated by integral membrane proteins. Integral membrane proteins of 35 (38) and 42 kD (26) have been detected by photoatiinity labeling with nascent secretory chains 1. Abbreviations used in this paper: K-RM, salt-extracted microsomal membranes; SRP, signal recognition particle. and synthetic signal sequences respectively. These polypeptides are proposed to function as signal sequence receptors during transport of the nascent chain across the microsomal membrane (26, 38). Several experimental approaches demonstrated that the mammalian translocation apparatus can accommodate a wider variety of translocation substrates and function under less restrictive conditions than had been initially believed. Both the amino-terminal and carboxyl-terminal domains flanking an internal signal sequence in an in vitro-constructed fusion protein can be translocated across microsomal membranes in vitro (23). Transport of nascent polypeptides can be initiated late during synthesis in a cotranslational assay (2, 32) or after synthesis in a posttranslational assay provided that the nascent chain remains bound to the ribosome via tRNA (22, 24). Although posttranslational membrane insertion of nascent secretory or membrane proteins can proceed by a guanine ribonucleotide-dependent reaction when the length of the nascent chain is relatively short (6, 15, 39), integration or translocation of larger nascent polypeptides requires ribonucleotide hydrolysis (21, 24). These observations have raised crucial questions concerning the mechanism of nascent chain transport across the mammalian endoplasmic reticulum. Are nascent polypeptides transported in a linear conformation when microsomal membranes are present during translation of the polypeptide, or are partially folded protein domains cotranslationally transported in a discontinuous © The Rockefeller University Press, 0021-9525/89/02/299/9 $2.00 The Journal of Cell Biology, Volume 108, February 1989 299-307 299 on M ay 2, 2017 D ow nladed fom Published February 1, 1989