Microinjection of ubiquitin: changes in protein degradation in HeLa cells subjected to heat-shock

Journal Article · Sun Mar 01 04:00:00 EST 1987 · J. Cell Biol.; (United States)

DOI:https://doi.org/10.1083/jcb.104.3.547· OSTI ID:6863407

Carlson, N; Rogers, S; Rechsteiner, M

Ubiquitin was radiolabeled by reaction with /sup 125/I-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.

• Research Organization: Univ. of Utah, Salt Lake City
• OSTI ID: 6863407
• Journal Information: J. Cell Biol.; (United States), Journal Name: J. Cell Biol.; (United States) Vol. 3; ISSN JCLBA
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANTI-INFECTIVE AGENTS
ANTIBIOTICS
AUTORADIOGRAPHY
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CHEMICAL REACTIONS
CYCLOHEXIMIDE
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENERGY
ERYTHROCYTES
FUNGICIDES
HEAT
HELA CELLS
HISTONES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KINETICS
LABELLING
MATERIALS
MOLECULAR WEIGHT
NUCLEI
NUCLEOPROTEINS
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PEPTIDES
PESTICIDES
POLYPEPTIDES
PROTEINS
PROTEOLYSIS
RADIOISOTOPES
REACTION KINETICS
TEMPERATURE EFFECTS