Dynamics of Ubiquitin Conjugation during Heat‐Shock Response Revealed by using a Monoclonal Antibody Specific to Multi‐Ubiquitin Chains

Abstract

Levels of intracellular multi-ubiquitinated proteins in heat-shocked HeLa cells were investigated using a monoclonal antibody specific to multi-ubiquitin chains. After heat-shock treatment at 42-44 "C for 30 min, the level of multi-ubiquitinated proteins increased within the first 2 h at 37°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 case for the level of ubiquitinated histone H2A. Immunocytochemical analysis 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 change in the activity of the 26s proteasome, but it did induce a gradual increase in activity of the ubiquitinating enzyme system. These results 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 and that the heat-shock-induced accumulation of multiubiquitinated proteins is caused by activation of the ubiquitinating enzyme system rather than inactivation of the 26s proteasome.Keywords: heat shock; proteasome; multi-ubiquitin chain; histone 2A; ubiquitin.A small set of universally conserved genes is activated when an organism is exposed to stress such as heat shock, heavy metals, ischemia, and irradiation [l-31. The proteins encoded by these genes are called stress proteins or heat-shock proteins. These proteins play key roles not only in the stress response, but also in the normal physiological response without stress [4].Ubiquitin, a heat-shock protein comprising 76 amino acid residues, is highly conserved and widely distributed in eukaryotes [5, 61. The ubiquitin gene contains a heat-shock promoter and is expressed by heat shock in eukaryotic cells such as chicken fibroblasts [7-91 and yeasts [lo, 111. ligase). Multi-ubiquitinated proteins produced in this way are degraded in an ATP-dependent manner by the 26s proteasome, a ubiquitin/ATP-dependent protease complex [see reviews 6, 27 -291.Although ubiquitin acts as a signal for protein degradation, several intracellular proteins that are not destined to be degraded are also ubiquitinated. The most abundant proteins are ubiquitinated histone H2A and H2B [30]. Approximately 10% of H2A is ubiquitinated at a ratio of one histone molecule to less than five ubiquitin molecules, and 2% of H2B is ubiquitinated at a ratio of one to less than four [31, 321. However, it is unclear whether these ubiquitin molecules are attached to histones via the multi-ubiquitin chain or not. Moreover, it is still uncertain why and how ubiquitinated histones are deubiquitinated in response to heat shock [33, 341 and in chromosome condensation during the mitotic cell cycle [35].When cells are exposed to heat shock, many aberrant proteins are produced from heat-sensitive proteins, nascent polypeptides, or partially ...

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