© The Rockefeller University Press,
0021-9525/1998//247 $5.00
The Journal of Cell Biology, Volume 140, Number 2,
, 1998 247-258
A Novel Fission Yeast Gene, tht1+, Is Required for the Fusion of Nuclear Envelopes during Karyogamy
Yoshie Tange*,
Tetsuya Horio*,
Mizuki Shimanuki*,
Da-Qiao Ding
,
Yasushi Hiraoka
, and
Osami Niwa*
* Kazusa DNA Research Institute, Kisarazu, Chiba 292, Japan; and the
Kansai Advanced Research Center, Communications Research Laboratory, Kobe 651-24, Japan
We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1+ gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1+ gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei.
Abbreviations used in this paper: DAPI, 4,6-diamidino-2-phenylindole; EDTA, ethylenediaminitetraacetate; endoH, endoglycosilase H; FISH, fluorescent in situ hybridization; GFP, green fluorescent protein; NSF, N-ethylmaleimide-sensitive factor; ORF, open reading frame; SPB, spindle pole body.
Dr. Horio present address is School of Medicine, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, Tokushima 770, Japan.

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