Yeast tom1 mutant exhibits pleiotropic defects in nuclear division, maintenance of nuclear structure and nucleocytoplasmic transport at high temperatures.
A tom1-1 mutant was isolated from Saccharomyces cerevisiae. At high temperatures, 60% of the cells were arrested as dumbbell forms with a single large nucleus containing duplicated DNA and a short spindle. Electron-microscopy showed electron-dense structures scattered within the nucleus. Indirect immunofluorescent microscopy revealed these structures to be fragmented nucleoli since the dotted structures were stained with anti-Nop1(fibrillarin) antibody in large regions of the nuclei. Fluorescent in situ hybridization analysis using oligo(dT) revealed nuclear accumulation of poly(A)+RNA. We cloned TOM1 which encodes a large protein (380kDa) with a hect (homologous to E6-AP C terminus)-domain at its C terminus. Deletions of either this hect-region or the entire gene made cellular growth temperature-sensitive. Site-directed mutagenesis of the conserved cysteine residue (tom1C3235A) in the hect-domain, supposed to be necessary for thioester-bond formation with ubiquitin, abolished the gene function. When a functional glutathione S-transferase (GST)-tagged hect protein was overproduced, it facilitated the protein conjugation with a myc-tagged ubiquitinRA, while this was not seen when GST-hectC3235A was overproduced. The protein conjugation with a hemagglutinin-tagged Smt3 was not affected by the overproduction of GST-hect. Taken together, we suggest that Tom1 is a ubiquitin ligase. As a multi-copy suppressor of tom1, we isolated STM3/NPI46/FPR3 which encodes a nucleolar nucleolin-like protein. We discuss possible functions of Tom1 with respect to the pleiotropic defects of nuclear division, maintenance of nuclear structure, and nucleocytoplasmic transport.
Utsugi
T
T
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
Hirata
A
A
Sekiguchi
Y
Y
Sasaki
T
T
Toh-e
A
A
Kikuchi
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D002455:Cell Division / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D016195:G2 Phase; D018628:Gene Dosage; D005786:Gene Expression Regulation; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D008025:Ligases / Q000378:metabolism; D008938:Mitosis; D009154:Mutation; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
D005656:Fungal Proteins; D018398:Homeodomain Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C115246:TOM1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D019696:Peptidylprolyl Isomerase; D008025:Ligases