Cotranslational Assembly Research Articles

Cotranslational assembly of myosin heavy chain in developing cultured skeletal muscle.

To examine how nascent myosin heavy chains associate with the cytoskeletons of developing muscle cells, we used pulse labeling, cell fractionation, and immunoprecipitation. More than 80% of nascent myosin heavy chains associate with the cytoskeleton. More than one-third of these nascent chains are not released by puromycin and/or RNase. The fraction of nascent heavy chains that resists release increases during development of muscle cells in culture. Treatment with cytochalasin D but not nocodazole decreases myosin heavy chain cotranslational assembly. These results indicate that (i) cotranslational assembly of myosin heavy chains is developmentally regulated, (ii) structures containing actin and not microtubules may mediate initial association of the heavy chains with the cytoskeleton, and (iii) the site of translation dictates where a significant fraction of the heavy chains will be inserted into the cytoskeleton.

Membrane assembly: Posttranslational insertion of M13 procoat protein into E. coli membranes and its proteolytic conversion to coat protein in vitro

The major coat protein (gene 8 product) of bacteriophage M13 is an integral membrane protein during infection of host cells. It is synthesized as a larger precursor (procoat) with a leader sequence of 23 amino acids at its amino terminus. In vivo studies have shown that procoat only inserts into the host-cell plasma membrane after its synthesis is completed. We now demonstrate that procoat can post-translationally insert into inverted cytoplasmic membrane vesicles from E. coli and can be processed proteolytically to yield coat protein. Procoat changes from an assembly-competent substrate to an incompetent (denatured) form within minutes after its synthesis; much of the procoat that accumulates during an hour of in vitro synthesis is therefore denatured. These studies emphasize the importance of stringent criteria for the demonstration of obligate cotranslational assembly.