Characterization of a Fission Yeast SUMO-1 Homologue, Pmt3p, Required for Multiple Nuclear Events, Including the Control of Telomere Length and Chromosome Segregation

Abstract

Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. Here we describe the identification of the SUMO-1 homologue of fission yeast, which we show to be required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of the pmt3 ؉ gene, the Schizosaccharomyces pombe homologue of SMT3, was not lethal, but mutant cells carrying the disrupted gene grew more slowly. The pmt3⌬ cells showed various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, we found that pmt3 ؉ is required for telomere length maintenance. Loss of Pmt3p function caused a striking increase in telomere length. When Pmt3p synthesis was restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase-and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation.Ubiquitin is a small (76-residue), abundant protein conserved in all eukaryotic cells. It exists in several cellular compartments, such as the cytosol, nucleus, and cell surface. It is well known that ubiquitin regulates the function and stability of target proteins through its posttranslational conjugation to target proteins. Before conjugation to target proteins, ubiquitin must be processed by a C-terminal hydrolase. The first step of the ubiquitin conjugation pathway is the ATP-dependent formation of a thioester bond between the conserved C-terminal glycine of processed ubiquitin and the active-site cysteine residue of an E1 ubiquitin-activating enzyme. The second step is the transfer of activated ubiquitin to the active-site cysteine of an E2 ubiquitin-conjugating enzyme. In the final step, the E2 enzyme may cooperate with an E3 ubiquitin protein ligase to form an isopeptide bond between the C-terminal glycine of ubiquitin and the ε-amino groups of lysine residues of target proteins. Ubiquitin covalently conjugated to target proteins can be removed by a ubiquitin isopeptidase (89).Recently, a number of novel ubiquitin-like proteins were independently discovered in a number of species, suggesting that ubiquitin is part of a family of related proteins involved in the covalent modification of proteins. The first example of such a protein was the 15-kDa interferon-inducible, ubiquitin crossreacting protein UCRP (25). UCRP contains two ubiquitinr...

SUMO-1 Modification Alters ADAR1 Editing Activity

“…To further characterize the subcellular distribution of SUMOconjugated proteins, a fusion of SUMO-1 to the green fluorescence protein (GFP-SUMO-1) was transiently expressed in HeLa cells. GFP-SUMO-1 can replace endogenous SUMO (pmt3) in fission yeast, suggesting that the GFP tag does not interfere with SUMO-1 function in vivo (Tanaka et al, 1999). Analysis of HeLa cells expressing GFP-SUMO-1 reveals nuclear staining with accumulation at the nuclear pore complexes and in nuclear bodies, as previously described (Figure 1A, arrows and arrowheads).…”

Ubc9 Regulates Mitosis and Cell Survival during Zebrafish Development

“…In budding yeast (Saccharomyces cerevisiae), Ubc9 deficiency is lethal, whereas analyses with a temperature-sensitive allele indicate that Ubc9 is required for progression through mitosis, mediating degradation of mitotic cyclins (Seufert et al, 1995). In contrary, Ubc9 (Hus5) mutants in fission yeast (Schizosaccharomyces pombe) are viable, but display defects during chromosome segregation and reduced cellular growth (al-Khodairy et al, 1995), similar to the defects of mutants in the Sumo gene (Tanaka et al, 1999). RNAi-mediated knockdown of Ubc9 in the nematode Caenorhabditis elegans results in several specific developmental defects that resemble phenotypes produced by mutations in known developmental regulators such as Hox genes (Jones et al, 2002).…”

“…This ubc9.1 expression pattern is in line with a role during cell proliferation in zebrafish larvae and with functional data obtained in other systems. Thus, Ubc9 has been implicated in various aspects of cell cycle control like chromosome condensation and segregation (Tanaka et al, 1999;Azuma et al, 2003) and the regulation of cyclin stability (Seufert et al, 1995;Dieckhoff et al, 2004), as well as in different DNA repair mechanisms (Hardeland et al, 2002;Hoege et al, 2002;Stelter and Ulrich, 2003).…”

The C-terminal zinc finger of the catalytic subunit of DNA polymerase is responsible for direct interaction with the B-subunit

“…The possibility that the slower mobility form might be either mono-ubiquitinated or sumoylated was also examined, by immunoblotting using anti-ubiquitin and anti-®ssion yeast SUMO (Pmt3) antisera (31,32). However, no cross-reaction could be detected, suggesting that V5-Pol3 is not modi®ed by either ubiquitin or SUMO.…”