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 reconstitu tion of nuclei and analysis of nuclear transport. Such cell-free extra cts contain three major N-acetylglucosaminylated proteins: p200, p97, and p60. Both p200 and p60 have been found to be components of the nuc lear pore. Here, the role of p97 has been investigated. Xenopus p97 wa s isolated and antisera were raised and affinity purified. Immunolocal ization experiments indicate that p97 is present in a punctate pattern on the nuclear envelope and also in the nuclear interior. Peptide seq uence analysis reveals that p97 contains a GLFG motif which defines a family of yeast nuclear pore proteins, as well as a peptide that is id entical 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 monoclo nal antibody to the GLFG domain cross-reacts with a major Xenopus prot ein of 97 kD and polyclonal antiserum to p97 recognizes the yeast GLFG nucleoporin family. The p97 antiserum was used to immunodeplete Xenop us egg cytosol and p97-deficient nuclei were reconstituted. The p97-de pleted nuclei remained largely competent for nuclear protein import. H owever, in contrast to control nuclei, nuclei deficient in p97 fail to grow in size over time and do not replicate their chromosomal DNA. ss DNA replication in such extracts remains unaffected. Addition of the N -acetylglucosaminylated nuclear proteins of Xenopus or rat reverses th ese replication and growth defects. The possible role(s) of p97 in the se nuclear functions is discussed.