Kap104p: A karyopherin involved in the nuclear transport of messenger RNA binding proteins

Science

Volumn 274, Issue 5287, 1996, Pages 624-627

  Aitchison, John D ^a   Blobel, Günter ^a   Rout, Michael P ^a  

^a ROCKEFELLER UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BINDING PROTEIN; CARRIER PROTEIN; FUNGAL PROTEIN; MESSENGER RNA; NUCLEOPORIN; NUCLEOPROTEIN; POLYADENYLATED RNA; RNA BINDING PROTEIN;

ARTICLE; CELL FRACTIONATION; CELL MEMBRANE TRANSPORT; NONHUMAN; PRIORITY JOURNAL; PROTEIN LOCALIZATION; PROTEIN PURIFICATION; PROTEIN TRANSPORT; RNA BINDING; SACCHAROMYCES CEREVISIAE; TEMPERATURE SENSITIVE MUTANT; YEAST;

AMINO ACID SEQUENCE; BIOLOGICAL TRANSPORT; CARRIER PROTEINS; CELL NUCLEUS; CYTOSOL; FUNGAL PROTEINS; KARYOPHERINS; MEMBRANE PROTEINS; MOLECULAR SEQUENCE DATA; MUTATION; NUCLEAR ENVELOPE; NUCLEAR PORE COMPLEX PROTEINS; NUCLEAR PROTEINS; NUCLEOCYTOPLASMIC TRANSPORT PROTEINS; RECOMBINANT FUSION PROTEINS; RNA, MESSENGER; RNA-BINDING PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; TEMPERATURE;

SACCHAROMYCES CEREVISIAE;

EID: 0029845118     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5287.624     Document Type: Article

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42. Kap104p; YBM7/YBR107c, position 270,905 to 273,658, chromosome II; Kap123p:YEU0/YER110c, position 376,750 to 380,088, chromosome V; Kap121p; PSE1/YMR308c, position 888,955 to 889,221, chromosome XIII.
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44. M. A. Andrade and P. Bork, Nature Genet. 11, 115 (1995). These structural domains are predicted to be antiparallel, amphipathic αhelices that form rod-like, tightly packed helical structures and present binding sites for additional proteins in loops between the packed helices. HEAT motifs have been predicted in mammalian karyopherin β (amino acids 122 to 482 and 600 to 725), Kap95p (amino acids 319 to 533 and 592 to 815), and Kap104p (amino acids 118 to 355 and 417 to 621), as well as in several other proteins, including Kap 123p and Kap 121 p. The predicted exposed loops of four adjacent proposed HEAT repeats of karyopherin β provide overlapping binding sites for karyopherin α and RanGTP [J. Morianu, G. Blobel, A. Radu, Proc. Natl. Acad. Sci. U.S.A. 93, 7059 (1996)]. Although the acidic amino acids of the central RanGTP binding site of karyopherin β (amino acids 333 to 340) are conserved in Kap95p (amino acids 338 to 344), the acidic stretch of Kap104p, which is aligned in the primary structure, is not present within its predicted HEAT motifs.
45. M. A. Andrade and P. Bork, Nature Genet. 11, 115 (1995). These structural domains are predicted to be antiparallel, amphipathic αhelices that form rod-like, tightly packed helical structures and present binding sites for additional proteins in loops between the packed helices. HEAT motifs have been predicted in mammalian karyopherin β (amino acids 122 to 482 and 600 to 725), Kap95p (amino acids 319 to 533 and 592 to 815), and Kap104p (amino acids 118 to 355 and 417 to 621), as well as in several other proteins, including Kap 123p and Kap 121 p. The predicted exposed loops of four adjacent proposed HEAT repeats of karyopherin β provide overlapping binding sites for karyopherin α and RanGTP [J. Morianu, G. Blobel, A. Radu, Proc. Natl. Acad. Sci. U.S.A. 93, 7059 (1996)]. Although the acidic amino acids of the central RanGTP binding site of karyopherin β (amino acids 333 to 340) are conserved in Kap95p (amino acids 338 to 344), the acidic stretch of Kap104p, which is aligned in the primary structure, is not present within its predicted HEAT motifs.
46. Deletion and disruption of the KAP104 gene was accomplished by replacement of the KAP104 open reading frame with the URA3-selectable marker in the diploid strain DF5 (38) by integrative transformation [R. Rothstein, Methods Enzymol. 194, 281 (1991)] with modifications as described (38). Heterozygous diploids containing a wild-type and a disrupted copy of KAP104 were sporulated, and tetrads were dissected on YPD plates at 23°C. Although all four spores could be recovered, haploid cells carrying a disrupted copy of KAP104 failed to germinate at 30°C, were severely impaired in their growth at 23°C, and died when transferred to 30°C.
47. Indirect immunofluorescence was performed after 8-min fixation of yeast spheroplasts in 3.7% formaldehyde as described (28), with mouse antiserum to purified Kap104p or Nab2p, monoclonal antibodies to Nsr1p and Nop1p, or rabbit antiserum to Nsp1p. Double immunofluorescent labeling was done with donkey DTAF [(dichlorotriazinyl)aminofluorescein]-conjugated antiserum to rabbit IgG and donkey Cy3-conjugated antiserum to mouse IgG. Composite images were collected as described (26).
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54. The protein A gene was integrated into thé genomic copy of each KAP gene, yielding functional chimeras as the only copy of each gene as described (38) (these strains are referred to as KAP95-A and KAP104-A). No growth defects were observed as a result of the presence of either tagged protein, and both subcellular fractionation and immunofluorescence microscopy of each of these constructs reflected the distribution observed with the wild-type proteins.
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58. The protein sequence of p68 and p70 yielded amino acids 295 to 312 of Nab2p and amino acids 245 to 257 of Nab4p or HRP1p (33). Nab2p YPD has the accession number YGL122c; HRP1p is also Nab4p (YPD accession number, YOL123w).
59. The KAP104 gene was cloned into PRS314, PRS316, and PRS317 to yield p104-TRP, p104-URA, and p104-LYS. p104-URA was transformed into diploid DF5 cells (38), carrying a deletion or disruption of one copy of KAP104 (kap104::ura3::HIS3). These cells were sporulated, tetrads dissected, and kap104::ura3::HIS3, p104-URA haploids were selected. These cells were then transformed with p104-TRP that had been passaged through the mutagenizing E. coli strain XL-1 Red. Transformants were transferred to 5-fluoroorotic acid to select against p104-URA at 23°C and then replica-plated at 30° and 37°C to identify temperature-sensitive strains. Strains that maintained their temperature sensitivity on YPD, and were rescued when retransformed with p104-LYS, were selected.
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63. N. Bonifaci and G. Blobel, personal communication.
64. J. D. Aitchison, M. P, Rout, M. Marelli, G. Blobel, R. W. Wozniak, J. Cell Biol. 131, 1133 (1995).
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66. We thank J. Fernandez and The Rockefeller University-Howard Hughes Medical Institute Biopolymer Facility for protein sequence analysis; S. Wente for Nup100p and Nup116p expression constructs; J. Kilmartin for antiserum to Nsr1p and J. Aris for antiserum to Nop1p; M. Rexach for many reagents and critically reading the manuscript; U. Nehrbass, F. Kessler, M. Rexach, the other members of the Blobel lab, and R. Wozniak for helpful discussions; and A. Antunez de Mayolo and E. Ellison for technical help. J.D.A. was supported by a postdoctoral fellowship from the Medical Research Council of Canada during a portion of this work.