Microinjection studies on selective protein degradation: relationships between stability, structure, and location.
We have used two approaches to study the relationship between the structure of a protein and its intracellular stability. First, over 35 proteins for which the primary and X-ray structure are known were radiolabeled and introduced into HeLa cells by RBC-mediated microinjection. We then measured the half-life and intracellular location of each protein. Analysis of these data has shown that the role of lysosomes in degradation of injected proteins is minor and nonselective, and that the subcellular location of a protein following injection may have a significant influence on its stability. Furthermore, no correlations were found between the half-life of a protein and its size, isoelectric point, hydrophobicity, or thermostability. However, initial multiple correlative analyses suggest that the stability of an injected protein may be related to an interplay between its location and the presence of unstable amino acids and disordered structure on the protein surface. Second, we have examined the primary sequences of 10 proteins with intracellular half-lives of less than 2 h. Each rapidly degraded protein contains at least one region of 12 to 45 amino acids rich in proline, glutamic acid, serine, and threonine (PEST). These PEST regions, flanked by positively charged amino acids, can also be identified by features common in their hydropathy plots. Similar inspection of 35 more stable, structurally characterized proteins revealed that only three contained weak PEST regions. On the basis of this information, we anticipated that caseins, which contain several PEST regions, would be rapidly degraded within eukaryotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)
D004795:Enzyme Stability; D005057:Eukaryotic Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008845:Microinjections; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014961:X-Ray Diffraction
D011506:Proteins