UMMS Affiliation

Department of Biochemistry

Publication Date

2-1-1989

Document Type

Article

Subjects

Biological Transport; Cell Membrane Permeability; Deoxycholic Acid; Detergents; Edetic Acid; Endopeptidase K; Intracellular Membranes; Membrane Glycoproteins; Microsomes; Molecular Weight; Octoxynol; Polyethylene Glycols; Prolactin; Protein Biosynthesis; Protein Precursors; Protein Sorting Signals; RNA, Messenger; Ribosomes; Serine Endopeptidases; Solubility; Vesicular stomatitis Indiana virus; Viral Envelope Proteins

Disciplines

Biochemistry | Cell Biology

Abstract

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.

Rights and Permissions

Citation: J Cell Biol. 1989 Feb;108(2):299-307.

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

The Journal of cell biology

PubMed ID

2537313

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.