Synthesis of intracellular membrane proteins in vitro. Relation between rough endoplasmic reticulum and mitochondrial outer membrane

Abstract

Hepatic rough microsomes were incubated in a messenger-dependent protein-synthesizing system from rabbit reticulocytes. Up to 30% of the total product labelled with [35S]methionine, and subsequently recovered with the microsomes, was located in an intrinsic protein fraction associated with these membranes, i.e. was retained by the membrane following extensive sonication in the presence of 1.5 M KCl, 0.1% deoxycholate, and 5 mM ethylenediaminetetra-acetate (EDTA). When products synthesized with the use of membrane-free mRNA from rough microsomes and free polysome were post-incubated with rough microsomes, ribosome-stripped rough microsomes, or outer mitochondrial membrane, low amounts of intrinsic-type polypeptide product were recovered with these membranes. Higher recovery was achieved, however, when ribosome-stripped rough microsomes were added at the beginning of polypeptide synthesis in a reticulocyte lysate supplemented with additional ribosomal-wash factors. Analysis of these products by polyacrylamide gel electrophoresis showed that a number co-migrated with intrinsic proteins located in both rough microsomes and mitochondrial outer membrane. In addition, a prominent in vitro product co-migrated with a major protein which is located in outer mitochondrial membrane fractions, but is barely detectable in rough microsomal fractions. The present experiments were unable to detect a unique set of intrinsic polypeptides which were synthesized and assembled in vitro under the direction of mRNA from free polysomes, and not from rough microsomes. The results suggest that synthesis of at least some intrinsic membrane proteins which are destined for the outer mitochondrial membrane occurs on rough ER in rat liver.