DYNAMICS OF UBIQUITIN CONJUGATION DURING HEAT-SHOCK RESPONSE REVEALEDBY USING A MONOCLONAL-ANTIBODY SPECIFIC TO MULTI-UBIQUITIN CHAINS

Levels of intracellular multi-ubiquitinated proteins in heat-shocked H eLa cells were investigated using a monoclonal antibody specific to mu lti-ubiquitin chains. After heat-shock treatment at 42-44 degrees C fo r 30 min, the level of multi-ubiquitinated proteins increased within t he first 2 h at 37 degrees C and returned to the initial level within the following 2 h. The accumulation of multi-ubiquitin conjugates was elevated by increasing the temperature, while the opposite was the cas e for the level of ubiquitinated histone H2A. Immunocytochemical analy sis revealed that the amount of ubiquitin conjugates rapidly increased in the cytosol and concomitantly decreased in the nucleus under heat- shock conditions. The heat-shock treatment elicited little apparent ch ange in the activity of the 26S proteasome, but it did induce a gradua l increase in activity of the ubiquitinating enzyme system. These resu lts strongly suggest that the level of cytoplasmic multi-ubiquitinated proteins and that of nuclear ubiquitinated histone H2A increases and decreases, respectively: in response to heat shock End that the heat-s hock-induced accumulation of multi-ubiquitinated proteins is caused by activation of the ubiquitinating enzyme system rather than inactivati on of the 26S proteasome.