Yeast tom1 mutant exhibits pleiotropic defects in nuclear division, maintenance of nuclear structure and nucleocytoplasmic transport at high temperatures

Gene. 1999 Jul 8;234(2):285-95. doi: 10.1016/s0378-1119(99)00197-3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Biological Transport
  • Cell Division / genetics
  • Cell Nucleus / metabolism*
  • Cell Nucleus / ultrastructure
  • Cytoplasm / metabolism*
  • Fungal Proteins / metabolism
  • G2 Phase
  • Gene Dosage
  • Gene Expression Regulation
  • Homeodomain Proteins / genetics*
  • Homeodomain Proteins / metabolism
  • Ligases / metabolism
  • Mitosis
  • Mutation
  • Peptidylprolyl Isomerase / genetics
  • Peptidylprolyl Isomerase / metabolism
  • RNA, Messenger / metabolism
  • Recombinant Fusion Proteins / genetics
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / ultrastructure
  • Saccharomyces cerevisiae Proteins*
  • Sequence Homology, Amino Acid
  • Temperature
  • Ubiquitin-Protein Ligases
  • Ubiquitins / metabolism

Substances

  • Fungal Proteins
  • Homeodomain Proteins
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Ubiquitins
  • TOM1 protein, S cerevisiae
  • Ubiquitin-Protein Ligases
  • Peptidylprolyl Isomerase
  • Ligases