A Conditional Lethal Mutant in the Fission Yeast 26 S Protease Subunit mts3+ Is Defective in Metaphase to Anaphase Transition

Abstract

We have isolated a conditional lethal mutant mts3 in the fission yeast Schizosaccharomyces pombe which at the permissive temperature is resistant to the mitotic poison MBC and at the restrictive temperature is defective in metaphase to anaphase transition. The predicted amino acid sequence of mts3 ؉ is 36% identical with the budding yeast gene NIN1. NIN1 cloned into a fission yeast expression vector can rescue both mts3 temperature-sensitive and null alleles demonstrating that NIN1 is the budding yeast homologue of the fission yeast mts3 ؉ gene. The phenotype of the mts3 null is identical with the mts3 ts mutant demonstrating that the phenotype of the mts3 ts mutant is due to loss of mts3 ؉ function. The deduced amino acid sequences of both mts3 ؉ and NIN1 show homology to peptide sequences obtained from subunit 14 of the 26 S protease purified from bovine or human cells.It is becoming increasingly recognized that the stability of proteins is an important factor in the regulation of gene expression. The main nonlysosomal intracellular proteolysis pathway in the cell is the ubiquitin pathway. The ubiquitin pathway has been implicated in the instability of a number of important regulatory proteins such as p53, c-Myc, c-Mos, and the mitotic cyclins. Ubiquitin is a 76-amino acid protein, that when covalently attached to a lysine residue of a protein, targets it for destruction (reviewed in Refs. 1-3).Biochemical analysis has determined that a multiprotein complex called the 26 S protease degrades proteins that have been targeted for destruction by the ubiquitin pathway. The 26 S protease is made up of two multiprotein functional components, the catalytic 20 S proteasome and the regulatory complex. The 20 S proteasome is a cylindrical structure composed of 14 different protein subunits and contains all the proteolytic activities of the complex. In vitro analysis on purified 20 S complexes has shown that proteins are degraded in a completely unregulated manner. Regulated proteolysis requires the addition of another multiprotein complex composed of at least another additional 15 different subunits to each end of the 20 S proteasome cylinder to form the 26 S protease. The different subunits of this regulatory complex are named according to their size on an SDS-polyacrylamide gel electrophoresis gel, S1 being the largest and S15 the smallest. With the formation of the 26 S protease, degradation of proteins is now carried out in a highly regulated manner. Certain substrates are degraded only if they have been polyubiquitinated and proteolysis is now ATP-dependent (4, 5).Recently, we isolated a conditional lethal mutant, mts2, in subunit 4 of the 26 S protease regulatory complex in the fission yeast Schizosaccharomyces pombe. Characterization of the phenotype of mts2 strain at the restrictive temperature demonstrated that the cells arrested at a particular point in the cell cycle, the metaphase stage of mitosis (6). In this paper, we describe another conditional mutant, mts3, which was isolated in the same screen. We show ...

The pad1 + Gene Encodes a Subunit of the 26 S Proteasome in Fission Yeast

“…Flow Cytometry-Flow cytometry was carried out using a Becton Dickinson FACScan using the method described previously (10,21).…”

“…In the presence of ATP, the 19 S cap binds to each end of the 20 S core and confers ATP dependence and specificity for ubiquitinated substrates on the 26 S complex (7). The budding yeast SUG1 gene (8) and the fission yeast mts2 ϩ gene (9) are known to encode ATPase components of the 19 S cap, while the fission yeast mts3 ϩ and mts4 ϩ genes encode non-ATPase subunits (10,11). At the restrictive temperature the phenotypes of the temperature-sensitive (ts) 1 mts2-1 and mts3-1 strains are transient cell cycle arrest at metaphase, indicating that the metaphase to anaphase transition is blocked (9,10).…”

“…The budding yeast SUG1 gene (8) and the fission yeast mts2 ϩ gene (9) are known to encode ATPase components of the 19 S cap, while the fission yeast mts3 ϩ and mts4 ϩ genes encode non-ATPase subunits (10,11). At the restrictive temperature the phenotypes of the temperature-sensitive (ts) 1 mts2-1 and mts3-1 strains are transient cell cycle arrest at metaphase, indicating that the metaphase to anaphase transition is blocked (9,10). This is similar to ts sug1-1 mutants that are unable to segregate their DNA and arrest after replication with an anucleate bud at the restrictive temperature (13).…”

Role of Hcn1 and Its Phosphorylation in Fission Yeast Anaphase-promoting Complex/Cyclosome Function

“…It binds Cut9 directly and is required to link different APC/C subcomplexes together. We have mapped the single site of phosphorylation within Hcn1 to Ser 48 , a consensus site for Cdk1. Mutational analysis indicated that Hcn1 phosphorylation is not an essential modification, but mutation of Ser 48 to alanine induced a novel S. pombe APC/C phenotype.…”

Mcs2 and a novel CAK subunit Pmh1 associate with Skp1 in fission yeast

“…We followed the levels of all three CAK subunits in Skp1 (skp1-3 [40]) or proteasome (mts3-1 [51]) temperature sensitive mutants, where known targets (Cdc18 and Cig2) of SCF-mediated ubiquitinylation and proteolysis accumulate at the restrictive temperature [52][53][54][55]. Total levels of HA-Mcs2, Pmh1-TAP, and Mcs6-TAP were followed by Western blot (anti-Ha or PAP; see Materials and methods) in wild type, skp1-3 or mts3-1 mutant strains after shift to restrictive temperature (Figs.…”