The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release.

Substrates enter the proteasome core particle (CP) through a channel that opens upon association with the regulatory particle (RP). Using yeast mutants, we show that channel opening is mediated by the ATPase domain of Rpt2, one of six ATPases in the RP. To test whether degradation products exit through this channel, we analyzed their size distribution. Their median length from an open-channel CP mutant was 40% greater than that from the wild-type. Thus, channel opening may enhance the yield of peptides long enough to function in antigen presentation. These experiments demonstrate that gating of the RP channel controls both substrate entry and product release, and is specifically regulated by an ATPase in the base of the RP.

Biochemical characterization of Caenorhabditis elegans UBC-1: self-association and auto-ubiquitination of a RAD6-like ubiquitin-conjugating enzyme in vitro.

The Caenorhabditis elegans ubiquitin-conjugating enzyme UBC-1 is distinct from other RAD6 homologues in possessing a C-terminal tail 40 amino acid residues long [Leggett, Jones and Candido (1995) DNA Cell Biol. 14, 883-891]. Such extensions from the core catalytic domain have been found in a subset of known conjugating enzymes, where they have been shown to have diverse roles including target recognition, membrane attachment and sporulation. In the present study we used mutagenesis in vitro to examine the role of the tail in specific aspects of UBC-1 structure and activity. Cross-linking experiments with purified recombinant UBC-1 reveal that it forms dimers and probably tetramers. The acidic tail of UBC-1 has an important role in this interaction because deletions of the tail significantly decrease, but do not abolish, this self-association. Ubiquitin conjugation assays show that, in addition to accepting a thiol-bound ubiquitin at its active site, UBC-1 is stably mono-ubiquitinated. Deletion analysis and site-directed mutagenesis localize the site of ubiquitination to Lys-162 in the tail. These findings demonstrate that the C-terminal tail of UBC-1 is important both for its quaternary structure and post-translational modification in vitro.

Caenorhabditis elegans UBC-1, a ubiquitin-conjugating enzyme homologous to yeast RAD6/UBC2, contains a novel carboxy-terminal extension that is conserved in nematodes.

The RAD6/UBC2 gene from Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme involved in DNA repair, induced mutagenesis, and sporulation. Here we report the isolation and characterization of the Caenorhabditis elegans RAD6 homolog designated ubc-1. Ubc-1 encodes a 21.5-kD protein that shares considerable identity with RAD6 (66%) as well as with other RAD6 homologs, including Schizosaccharomyces pombe rhp6+ (70%), Drosophila melanogaster Dhr6 (83%), and the two human homologs HHR6A and HHR6B (84% and 83%, respectively). However, UBC-1 is distinct in being the only known RAD6 homolog, other than RAD6 itself, with a carboxy-terminal extension. Analysis of UBC-1 homologs from C. briggsae and Ascaris suum show that the presence of the carboxy-terminal extension is conserved in nematodes. When constitutively expressed from the yeast promoter ADH1, ubc-1 complements the DNA repair functions in a S. cerevisiae rad6 delta mutant. Surprisingly, ubc-1 fails to complement the sporulation function of RAD6, despite its possession of an acidic carboxy-terminal tail. C. elegans UBC-1 is capable of forming a thiol-ester bond with ubiquitin, but, unlike RAD6, is unable to transfer ubiquitin to histone H2B in vitro. Both cis and trans splicing are involved in the maturation of the ubc-1 transcript. The presence of the SL2 trans-splice leader in the ubc-1 transcript suggests that ubc-1 may be co-transcribed as part of a polycistronic message.