Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication.
Xenopus egg extracts provide a powerful system for in vitro reconstitution of nuclei and analysis of nuclear transport. Such cell-free extracts contain three major N-acetylglucosaminylated proteins: p200, p97, and p60. Both p200 and p60 have been found to be components of the nuclear pore. Here, the role of p97 has been investigated. Xenopus p97 was isolated and antisera were raised and affinity purified. Immunolocalization experiments indicate that p97 is present in a punctate pattern on the nuclear envelope and also in the nuclear interior. Peptide sequence analysis reveals that p97 contains a GLFG motif which defines a family of yeast nuclear pore proteins, as well as a peptide that is identical at 11/15 amino acids to a specific member of the GLFG family, NUP116. An additional peptide is highly homologous to a second sequence found in NUP116 and other members of the yeast GLFG family. A monoclonal antibody to the GLFG domain cross-reacts with a major Xenopus protein of 97 kD and polyclonal antiserum to p97 recognizes the yeast GLFG nucleoporin family. The p97 antiserum was used to immunodeplete Xenopus egg cytosol and p97-deficient nuclei were reconstituted. The p97-depleted nuclei remained largely competent for nuclear protein import. However, in contrast to control nuclei, nuclei deficient in p97 fail to grow in size over time and do not replicate their chromosomal DNA. ssDNA replication in such extracts remains unaffected. Addition of the N-acetylglucosaminylated nuclear proteins of Xenopus or rat reverses these replication and growth defects. The possible role(s) of p97 in these nuclear functions is discussed.
Powers
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Department of Biology 0347, University of California at San Diego, La Jolla 92093-0347.
Macaulay
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Masiarz
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Forbes
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DJ
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus / Q000502:physiology; D002875:Chromosomes / Q000378:metabolism; D004261:DNA Replication; D005260:Female; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D016601:RNA-Binding Proteins; D012091:Repetitive Sequences, Nucleic Acid; D029701:Saccharomyces cerevisiae Proteins; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
D005656:Fungal Proteins; D008565:Membrane Proteins; C077731:NUP100 protein, S cerevisiae; C077730:NUP116 protein, S cerevisiae; C088627:NUP145 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C092347:nuclear pore protein p97