Membrane-associated protein synthesis in Streptococcus faecalis

Abstract

1. Cell-free extracts of Streptococcus faecalis were separated centrifugally into a ribosomal, a washed membrane, and several intermediate fractions. All fractions incorporate labeled amino acids into hot trichloroacetic acid-insoluble material. The requirements for amino acid incorporation are similar for all fractions, and electro-phoresis of the reaction products gives identical polypeptide peaks. 2. All particulate fractions contain phospholipid, and the level of amino acid incorporation is proportional to phospholipid content. No qualitative differences are found in the phospholipid composition of the various fractions. 3. Incorporation by cell fractions is also proportional to RNA content, except for the ribosomal fraction and a first membrane washing, where “excess” RNA appears to be present. On sucrose density gradients of ribosomal fractions the bulk of the “excess” RNA is isolated as a broad peak with a sedimentation coefficient of 80–150 S. On mild ribonuclease (EC 2.7.7.16) treatment this peak dissociates into 30-20 S ribosomal subunits. 4. In addition to large 30-20 S ribosomal aggregates, crude extracts of S. faecalis contain large polyribosomal aggregates. The polyribosomes, consisting of 30-S and 50-S subunits are released from membranes on exposure to deoxycholate. The ribosomes appear to be bound to membranes through the 50-S subunit. These membrane-bound polyribosomes are ribonuclease-sensitive, but are not degraded on incubation with a soluble fraction of S. faecalis. 5. It is concluded that in S. faecalis the bulk of the ribosomes are in a high state of aggregation. In fractionated extracts, protein synthesis is a function of the concentration of membrane-associated polyribosomes. Possible implications of this finding on mechanisms of protein synthesis are discussed.