Microinjection of ubiquitin: changes in protein degradation in HeLa cells subjected to heat-shock.
Ubiquitin was radiolabeled by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using erythrocyte-mediated microinjection. The injected cells were then incubated at 45 degrees C for 5 min (reversible heat-shock) or for 30 min (lethal heat-shock). After either treatment, there were dramatic changes in the levels of ubiquitin conjugates. Under normal culture conditions, approximately 10% of the injected ubiquitin is linked to histones, 40% is found in conjugates with molecular weights greater than 25,000, and the rest is unconjugated. After heat-shock, the free ubiquitin pool and the level of histone-ubiquitin conjugates decreased rapidly, and high molecular weight conjugates predominated. Formation of large conjugates did not require protein synthesis; when analyzed by two-dimensional electrophoresis, the major conjugates did not co-migrate with heat-shock proteins before or after thermal stress. Concomitant with the loss of free ubiquitin, the degradation of endogenous proteins, injected hemoglobin, BSA, and ubiquitin was reduced in heat-shocked HeLa cells. After reversible heat-shock, the decrease in proteolysis was small, and both the rate of proteolysis and the size of the free ubiquitin pool returned to control levels upon incubation at 37 degrees C. In contrast, neither proteolysis nor free ubiquitin pools returned to control levels after lethal heat-shock. However, lethally heat-shocked cells degraded denatured hemoglobin more rapidly than native hemoglobin and ubiquitin-globin conjugates formed within them. Therefore, stabilization of proteins after heat-shock cannot be due to the loss of ubiquitin conjugation or inability to degrade proteins that form conjugates with ubiquitin.
D001345:Autoradiography; D003513:Cycloheximide / Q000494:pharmacology; D004912:Erythrocytes / Q000502:physiology; D006367:HeLa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D006657:Histones / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008845:Microinjections; D009363:Neoplasm Proteins / Q000096:biosynthesis; D013462:Sulfur Radioisotopes; D014452:Ubiquitins / Q000008:administration & dosage / Q000378:metabolism*
D006360:Heat-Shock Proteins; D006657:Histones; D009363:Neoplasm Proteins; D013462:Sulfur Radioisotopes; D014452:Ubiquitins; D003513:Cycloheximide