Segregation of the Polypeptide Translocation Apparatus to Regions of the Endoplasmic Reticulum Containing Ribophorins and Ribosomes. I. Functional Tests on Rat Liver Microsomal Subfractions

• Published in: The Journal of cell biology Vol. 99; no. 6; pp. 2247 - 2253
• Main Authors: Amar-Costesec, Alain, Todd, John A., Kreibich, Gert
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.12.1984; The Rockefeller University Press
• Subjects: Animals; Biological and medical sciences; Cell Fractionation; Cell membranes. Ionic channels. Membrane pores; Cell structures and functions; Centrifugation; Cytochromes; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - ultrastructure; Female; Fundamental and applied biological sciences. Psychology; Liver; Liver - metabolism; Liver - ultrastructure; Membrane Proteins - metabolism; Microscopy, Electron; Microsomes; Microsomes, Liver - metabolism; Microsomes, Liver - ultrastructure; Molecular and cellular biology; Peptides - genetics; Polyribosomes; Protein Processing, Post-Translational; Protein transport; proteins; Rats; Rats, Inbred Strains; ribophorin; Ribosomes; Ribosomes - metabolism; Ribosomes - ultrastructure; RNA; rough endoplasmic reticulum; Proteins; Ribosome; Secretion; Membrane transport; Rough microsome; Localization; Endoplasmic reticulum
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.99.6.2247

A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate that the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle.