Background: Recent cDNA cloning of two homologous proteasome activators, PA28a and PA28b, indicated the presence of a structurally related third protein, Ki antigen, but a functional relationship between Ki antigen and the two PA28 proteins is unknown. Accumulating evidence has implicated an important role for PA28 in the major histocompatibility complex (MHC) class I-restricted antigen processing pathway. Recently, an immunomodulatory cytokine g-interferon (g-IFN) was found to increase greatly the messages for PA28a and PA28b, but not Ki antigen, in human cells.

The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome.

The nucleotide sequence of a cDNA that encodes a new regulatory subunit, named ~45, of the 26s proteasome of human hepatoblastoma HepG2 cells has been determined. The polypeptide predicted from the open reading frame consists of 406 amino acid residues with a calculated molecular weight of 45770 and isoelectric point of 8.35. The sequences of several fragments of bovine ~45, determined by protein chemical analyses, spanning 27% of the complete structure, were found to be in excellent accord with those deduced from the human cDNA sequence. Computer analysis showed that ~45 belongs to a family of putative ATPases which includes regulatory components of 26s proteasomes. The overall structure of ~45 was found to be homologous to that of yeast Suglp, which has been identified as a transcripional factor. It is closely similar, but not identical to the sequence reported for Tripl, a functional homolog of Suglp in human tissues. These results are consistent with the possibility that Sugl-like proteins with distinct sequence function in transcription and protein degradation in human cells. However, the alternative hypothesis, that the same gene locus encodes both ~45 and Tripl, cannot be excluded on the basis of such closely similar sequences. In either case, both proteins are likely to function equivalently well in either transcription or protein degradation.