Rogers S W, Rechsteiner M
Department of Biology, University of Utah, Salt Lake City 84132.
J Biol Chem. 1988 Dec 25;263(36):19843-9.
Thirty-five proteins of known x-ray structure were radioiodinated and injected into HeLa cells. The cells were then cultured in the presence or absence of the lysosomotropic agents, ammonium chloride and chloroquine. These compounds did not inhibit the degradation of an injected protein unless its half-life was greater than 45 h. Among the more stable proteins the extent of inhibition was proportional to their half-lives. These results indicate that all injected proteins are transferred to lysosomes at comparable rates such that the fraction of a specific protein degraded in lysosomes depends upon its rate of degradation in the cytosol. That is, basal autophagy is nonselective in HeLa cells. The intracellular location of each injected protein was measured by homogenization of injected cells in sucrose and differential sedimentation or by extraction in buffers containing Triton X-100. Solubilities of the injected proteins ranged from 6 to 89%, and stabilities of 10 proteins, originally extracellular in function, were inversely proportional to their solubility. These results illustrate the potential importance of subcellular location on protein stability in the cytosol.
对35种已知X射线结构的蛋白质进行放射性碘化标记，然后注射到HeLa细胞中。随后，将细胞在溶酶体促渗剂氯化铵和氯喹存在或不存在的情况下进行培养。除非注射蛋白质的半衰期大于45小时，否则这些化合物不会抑制其降解。在较稳定的蛋白质中，抑制程度与其半衰期成正比。这些结果表明，所有注射的蛋白质都以相当的速率转移到溶酶体中，因此在溶酶体中降解的特定蛋白质的比例取决于其在细胞质中的降解速率。也就是说，在HeLa细胞中基础自噬是非选择性的。通过将注射细胞在蔗糖中匀浆并进行差速沉降，或在含有Triton X-100的缓冲液中提取，来测量每种注射蛋白质的细胞内定位。注射蛋白质的溶解度范围为6%至89%，10种原本在细胞外起作用的蛋白质的稳定性与其溶解度成反比。这些结果说明了亚细胞定位对细胞质中蛋白质稳定性的潜在重要性。
