Translational initiation factor and ribosome association with the cytoskeletal framework fraction from HeLa cells

The association of mRNA and ribosomes with the cytoskeleton of eucaryotic cells may be important for protein synthesis and its regulation. HeLa cells were gently lysed with detergent, and soluble and cytoskeletal framework subfractions were prepared by centrifugation. We analyzed these fractions for ribosomes and confirmed earlier findings that polysomes are preferentially associated with the cytoskeletal fraction. The levels of initiation factors elF-2, elF-3, elF-4A, and elF-4B were quantitated by immunoblotting; all are enriched in the cytoskeletal fraction relative to the soluble fraction. Heat shock, fluoride, pactamycin, and cytochalasin caused the release of both ribosomes and initiation factors into the soluble fraction. However, treatment of the cytoskeletal fraction with EDTA or low levels of ribonuclease resulted in polysome degradation but no release. Therefore initiation factor association with the cytoskeletal framework correlates with the presence of ribosomes, whereas ribosome association does not require intact mRNA.     

Immunoblot analysis of the structure of protein synthesis initiation factor eIF3 from HeLa cells

Translational initiation factor eIF3 is a large, multisubunit protein complex involved in early steps of the initiation pathway. Affinity-purified polyclonal antibodies were used to analyze by immunoblotting the mass and charge characteristics of the subunits in HeLa cell lysates and in purified eIF3 preparations. The evidence indicates that eIF3 comprises at least seven antigenically distinct subunits: p170, p115, p66, p47, p44, p40, and p36. During purification, p170, p115, and p66 are partially degraded to smaller forms, which appear to be the major cause of variable subunit composition among preparations of eIF3.