Collagen synthesis on polyribosomes of cultured mammalian fibroblasts

Abstract

Ribosomes attached to the endoplasmic reticulum have been separated from those unattached to cytoplasmic membranes of cultured, stationary mammalian fibroblasts. In linear sucrose gradients, approximately one-half of the unattached ribosomes were recovered as 80 s particles, the rest as polyribosomes. The ribosomes freed by deoxycholate from the endoplasmic reticulum were recovered predominantly as polyribosomes. Free and attached polyribosomes had about the same size distributions in the gradients, were equally active in protein synthesis, and nascent collagen and non-collagen proteins were synthesized over all size distributions of polyribosomes. Polyribosomes sedimenting at 180 s were most abundant, and fractions sedimenting at 210 to 220 s were most active in collagen synthesis. There was no evidence in this system for appreciable synthesis of collagen or non-collagen protein on polyribosomes sedimenting faster than 330 s. Hydroxylation of prolyl residues in nascent collagen occurred while the peptide chains were attached to polyribosomes of the endoplasmic reticulum. Pulse-chase experiments indicated that as these nascent collagen chains neared completion, as judged by their hydroxyproline content, they moved with the fastest sedimenting polyribosomes. Release of completed collagen molecules presumably occurred from such units. Nascent collagen appeared to help stabilize polyribosomes of the endoplasmic reticulum.