CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

Abstract

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.

Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1.

“…Functional Relationship between NIN1 and SUN Genes and Their Roles in Cell Cycle Control In addition to the major defects of the ninl-l mutant at the G2-M and G1-S-phase boundaries, this mutant displays some other phenotypes such as instability of minichromosome maintenance (Kominami and Toh-e, 1994) and sensitivity to cadmium (Yokota et al, 1996). All of these phenotypes of the ninl-l mutant were suppressed by overexpressing either SUNI or SUN2 (our unpublished results).…”

“…For example, inactivation of SEN3, the largest subunit of the 26S proteasome, resulted in a temperature-sensitive phenotype but no cell cycle defect (Yokota et al, 1996). On the other hand, the temperature-sensitive ninl-l mutant and cim mutants arrested at the G2-M boundary, the G1-S-phase boundary, or both.…”

“…At present, it is widely believed that the ubiquitination determines the specificity of the degradation, where proteasome is constitutively active and degrades proteins marked by the ubiquitination. The facts that proteasome participates in degradation of various abnormal proteins produced under stress or in degradation via the N-end rule pathway (Yokota et al, 1996) favor the thought that proteasome is constitutively active. Nevertheless, there is some evidence that alterations in proteasome function are also subject to regulation.…”

“…Fortunately, the structure of the 26S proteasome is well conserved among eukaryotes. We and others identified several yeast genes which are homologues of the genes for human proteasome components: NIN1/p31 (Kominami et al, 1995), SEN3/p112 (DeMarini et al, 1995;Yokota et al, 1996), CIM5/MSS1 (Ghislain et al, 1993), and NAS1/p97 . These yeast mutants will afford a powerful experimental system to assign a function to each of the subunits of the regulatory component of the 26S proteasome.…”

The Assembly Pathway of the 19S Regulatory Particle of the Yeast 26S Proteasome

“…Next, we ask the following question: Are the lid and the base assembled independently to each other? To address this issue, we examined the status of lid assembly in a temperature-sensitive base mutant of RPN2 termed ⌬N rpn2 (equivalent to rpn2⌬ described by Yokota et al 1996). ⌬N rpn2 is a mutant that carries an N-terminal truncated (⌬1-220aa) version of RPN2 in a null rpn2 strain.…”