Amino acid incorporation into protein by ribosomes bound to chloroplast thylakoid membranes: formation of discrete products

Abstract

A system which incorporates amino acids into proteins of chloroplast membranes of Chlamydomonas reinhardti is described. It consists of chloroplast ribosomes bound to thylakoid membranes and cell extract. mRNA is present in this thylakoid-ribosome complex, since neither initiation nor RNA synthesis seems to be required for amino acid incorporation. Incorporation requires ATP, GTP and a soluble portion of cell extract. It is inhibited by chloramphenicol, but not cycloheximide. Most incorporated radioactivity remains bound to the membranes. Although a large portion of this labeled membrane-bound protein occurs as nascent polypeptides, a portion appears at least four products of discrete molecular weights. The major in vitro product migrates as a polypeptide of 23 000 daltons. We conclude that a portion of chloroplast membrane proteins is not only made within the chloroplast, but directly on the membranes. We had previously observed that release of membrane-bound ribosomes is partially dependent on puromycin, and concluded that some membrane-bound ribosomes were attached to the membranes through nascent protein chains. Thus, our results suggest that some chloroplast membrane proteins are inserted into the membranes as they are synthesized. This chloroplast membrane amino acid incorporation system offers a promising tool for studying biosynthesis of membrane proteins, and how they become inserted into chloroplast thylakoids to form functional membranes.