REGULATION OF INTRACELLULAR PROTEIN DEGRADATION

Cellular homeostasis is maintained by the dynamic balance of regulated expression of genes and the regulated degradation of proteins. The role of chromatin architecture in the regulation of gene expression was in the focus of our previous research activity. During the last years we concentrate on studying the structure and function of the 26S proteasome, a large proteolytic complex responsible for the regulated intracellular degradation of proteins.

Selective intracellular protein degradation proceeds in two steps: first the protein to be degraded is marked by a specific posttranslational modification (ubiquitination) performed by a series of ubiquitin conjugating enzymes, and this is followed by the proteolytic step performed by a very large multisubunit protease (26S proteasome), which cleaves only the ubiquitinated proteins. The 20S proteasome, a multicatalytic large protease complex was identified as the catalytic component of the 26S proteasome. The 20S proteasome alone has no specificity: it cleaves any non-ubiquitinated protein. The regulatory component of the 26S proteasome, responsible for the selective degradation of ubiquitinated proteins was identified, purified and characterized in my laboratory (J. Biol. Chem. 268, 9055. 1993). We showed that the 26S proteasome can be reconstituted in vitro from the purified regulatory complex and the 20S proteasome, and the in vitro reconstituted 26S proteasome degrades selectively only ubiquitinated proteins. The regulatory complex is a large (800 kDa) multiprotein particle. We have cloned and sequenced several subunits of this complex. Yeast homologs of two subunits of the regulatory complex were identified. Mutation of these subunits in S. cerevisiae stabilized the short-lived ubiquitin-proline-beta-galactosidase fusion protein and the CLB2 and CLB3 cyclins, and arrested cell division in G2/metaphase (Nature 366, 358. 1993). This was the first direct evidence that (i) the 26S proteasome is required for the in vivo degradation of ubiquitinated proteins, and (ii) cyclins are degraded in vivo by the ubiquitin-26S proteasome system. Recently we have identified, cloned and sequenced all the subunits of the Drosophila regulatory complex, and using a chemical cross-linking approach and a highly selective immunological detection system we have identified the closely-spaced subunits of the regulatory complex. To study the in vivo function of the multiubiquitin-binding subunit of the regulatory complex (subunit p54) we have deleted the single-copy gene of subunit p54. The homozygous Drosophila deletion results in pupal lethality, the large accumulation of multiubiquitinated proteins, severe mitotic defects and a co-ordinated upregulation of all the proteasomal genes. The function of the different domains of the multiubiquitin-binding subunit is analyzed recently by establishing transgenic Drosophila strains expressing the full-length subunit, its N-terminal half carrying the VWA domain or its C- terminal half carrying the UIM-domains. We have shown that the C-terminal half of the subunit in its extraproteasomal state is multiubiquitinated, the site of this modification is the cluster of lysines at the very C-terminal end of the subunit, and this modification is not a degradation signal. Recently we analyze the developmentally regulated changes of the 26S proteasomes. We found that the stoichiometry of the multiubiquitin-binding subunit drops suddenly at the end of the embryogenesis, remains low through the larval stages, starts to increase at the end of the third instar larval stage and reaches a high level in pupae, adults and embryos. A specific proteolytic system was identified which is responsible for the selective degradation of the ubiquitin-binding subunit during the larval stage. There is an inverse relationship between this larval-specific proteolytic system and the 26S proteasomes.