Degradation of proteins microinjected into IMR-90 human diploid fibroblasts.
Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte-mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.
D002459:Cell Fusion; D002478:Cells, Cultured; D003470:Culture Media; D004910:Erythrocyte Membrane; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007467:Iodoproteins / Q000378:metabolism; D007526:Isoelectric Point; D010447:Peptide Hydrolases / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship
D003470:Culture Media; D007467:Iodoproteins; D011506:Proteins; D010447:Peptide Hydrolases