We have used
proteinase K as a probe to detect cytoplasmically and luminally 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 behaviour 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.