Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum.

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Abstract
The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes [rat liver] was demonstrated under experimental conditions. High-salt-washed rough microsomes were treated with pancreatic RNase to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomes. Freeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4.degree. C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. When RNase-treated rough microsomes were brought to 24.degree. C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. The formation of ribosomal aggregates on the microsomal surface may result from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains and other associated membrane proteins. The inhibition of ribosome mobility after maintaining rough microsomes at 4.degree. C after RNase or antibody treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-.ANG. intramembrane particles in control rough microsomes. Aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.