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 tempera tures, 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 im munofluorescent microscopy revealed these structures to be fragmented nucle oli since the dotted structures were stained with anti-Nop1 (fibrillarin) a ntibody in large regions of the nuclei. Fluorescent in situ hybridization a nalysis 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 he ct-region or the entire gene made cellular growth temperature-sensitive. Si te-directed mutagenesis of the conserved cysteine residue (tom1(C3235A)) in the hect-domain, supposed to be necessary for thioester-bond formation wit h ubiquitin, abolished the gene function. When a functional glutathione S-t ransferase (GST)-tagged beet protein was overproduced, it facilitated the p rotein conjugation with a myc-tagged ubiquitin(RA), while this was not seen when GST-hect(C3235A) was overproduced. The protein conjugation with a hem agglutinin-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 stru cture, and nucleocytoplasmic transport. (C) 1999 Elsevier Science B.V. All rights reserved.