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 (380 kDa) 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 (tom1(C3235A)) 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 beet protein was overproduced, it facilitated the protein conjugation with a myc-tagged ubiquitin(RA), while this was not seen when GST-hect(C3235A) 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. (C) 1999 Elsevier Science B.V. All rights reserved.