A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes.
In a screen for Saccharomyces cerevisiae genes required for nucleocytoplasmic transport of messenger RNA, we identified the RAT7 gene (ribonucleic acid trafficking), which encodes an essential protein of 1,460 amino acids. Rat7p is located at the nuclear rim in a punctate pattern characteristic of nucleoporins. Furthermore, the central third of Rat7p contains 22 XXFG and three XFXFG degenerate repeats that are similar to signature GLFG and XFXFG repeats present in a majority of yeast and some mammalian nucleoporins sequenced to date. Shift of a strain bearing the temperature-sensitive rat7-1 allele from 23 degrees C to 37 degrees C resulted in rapid (within 15 minutes) cessation of mRNA export, but did not cause concomitant cytoplasmic accumulation of a reporter protein bearing a nuclear localization signal. This suggests that Rat7p may play a direct role in nucleocytoplasmic export of RNA. Immunofluorescence and thin section electron microscopy revealed that in rat7-1 cells grown at 23 degrees C, the majority of nuclear pore complexes (NPCs) were clustered on one side of the nucleus. No ultrastructural abnormalities of the nuclear envelope were seen. Interestingly, shifting rat7-1 cells to 37 degrees C for 1 h caused the NPCs to disperse, restoring near wild-type NPC distribution. After this temperature shift, the mutant Rat7p was no longer detectable by immunofluorescence.
Gorsch
L C
LC
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Dockendorff
T C
TC
Cole
C N
CN
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D015825:Chromosomes, Fungal / Q000235:genetics; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D005800:Genes, Fungal / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics; D017930:Genes, Reporter; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009685:Nuclear Envelope / Q000378:metabolism* / Q000648:ultrastructure; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA
D008565:Membrane Proteins; C093510:NUP159 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins