Glycosylation of nuclear pore protein p62. Reticulocyte lysate catalyzes O-linked N-acetylglucosamine addition in vitro.

Accurate inheritance of genomic content during cell division is dependent on synchronized changes in cellular organization and chromosome dynamics. Elucidating how these events are coordinated is necessary for a complete understanding of cell proliferation. Previous in vitro studies have suggested that the nuclear pore protein Nup153 is a good candidate for participating in mitotic coordination. To decipher whether this is the case in mammalian somatic cells, we reduced the levels of Nup153 in HeLa cells and monitored consequences on cell growth. Reduction of Nup153 resulted in a delay during the late stages of mitosis accompanied by an increase in unresolved midbodies. Depletion of Nup153 to an even lower threshold led to a pronounced defect early in mitosis and an accumulation of cells with multilobed nuclei. Although global nucleocytoplasmic transport was not significantly altered under these depletion conditions, the FG-rich region of Nup153 was required to rescue defects in late mitosis. Thus, this motif may play a specialized role as cells exit mitosis. Rescue of the multilobed nuclei phenotype, in contrast, was independent of the FG-domain, revealing two separable roles for Nup153 in the execution of mitosis.

Download Full-text

Sumoylation of Mdm2 by Protein Inhibitor of Activated STAT (PIAS) and RanBP2 Enzymes

Journal of Biological Chemistry ◽

10.1074/jbc.m208319200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 50131-50136 ◽

Cited By ~ 62

Author(s):

Yasuhiro Miyauchi ◽

Satomi Yogosawa ◽

Reiko Honda ◽

Tamotsu Nishida ◽

Hideyo Yasuda

Keyword(s):

Mammalian Cells ◽

Nuclear Translocation ◽

Nuclear Pore ◽

Intact Cells ◽

Mdm2 Protein ◽

Nuclear Pore Protein ◽

Lysine Residues ◽

Localization Signal ◽

Pore Protein

Mdm2, a ubiquitin ligase that acts on p53, is regulated by sumoylation. In the current study, we identify the enzymes responsible for the sumoylation of Mdm2. When mammalian cells are co-transfected with cDNAs encoding Mdm2 and PIAS1 or PIASxβ (proteininhibitor ofactivatedSTAT) as sumoylation enzymes, Mdm2 is highly sumoylated. Mdm2 is also sumoylated in anin vitrosystem containing PIASxβ, PIAS1, and RanBP2. When several lysine residues of Mdm2 were sequentially mutated to arginine, the K182R mutant was not sumoylated in intact cells; however, in thein vitrosystem this mutant was sumoylated by PIAS1, PIASxβ, and RanBP2 as efficiently as the wild-type Mdm2 protein. Lysine residues 182 and 185 map within the nuclear localization signal of Mdm2. A K185R mutant of Mdm2 is sumoylated in intact cells, whereas a K182R protein is not. Only a Mdm2 protein bearing the K182R mutation is localized exclusively in the cytoplasm. Because RanBP2 is a nuclear pore protein and PIAS proteins are localized within the nucleus, our data suggest that Mdm2 is sumoylated during nuclear translocation by RanBP2 and then further sumoylated once in the nucleus by PIASxβ and PIAS1.

Download Full-text

Reconstitution of nuclear protein transport with semi-intact yeast cells.

The Journal of Cell Biology ◽

10.1083/jcb.123.4.785 ◽

1993 ◽

Vol 123 (4) ◽

pp. 785-798 ◽

Cited By ~ 48

Author(s):

G Schlenstedt ◽

E Hurt ◽

V Doye ◽

P A Silver

Keyword(s):

Nuclear Envelope ◽

Protein Transport ◽

Protein Import ◽

Nuclear Protein ◽

Yeast Cells ◽

Nuclear Pore ◽

Nuclear Pore Protein ◽

Nuclear Protein Import ◽

Pore Protein

We have developed an in vitro nuclear protein import reaction from semi-intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mammalian cells, a protein containing the nuclear localization sequence (NLS) of SV40 large T-antigen is transported into the nucleus. Proteins with mutant NLSs are not imported. In the absence of cytosol, binding of NLS-containing proteins occurs at the nuclear envelope. N-ethylmaleimide treatment of the cytosol as well as antibodies to the nuclear pore protein Nsp1 inhibit import but not binding to the nuclear envelope. Yeast mutants defective in nuclear protein transport were tested in the in vitro import reaction. Semi-intact cells from temperature-sensitive nsp1 mutants failed to import but some binding to the nuclear envelope was observed. On the other hand, no binding and thus no import into nuclei was observed in semi-intact nsp49 cells which are mutated in another nuclear pore protein. Np13 mutants, which are defective for nuclear protein import in vivo, were also deficient in the binding step under the in vitro conditions. Thus, the transport defect in these mutants is at the level of the nucleus and the point at which nuclear transport is blocked can be defined.

Download Full-text

Introgression of Drosophila simulans Nuclear Pore Protein 160 in Drosophila melanogaster Alone Does Not Cause Inviability but Does Cause Female Sterility

Genetics ◽

10.1534/genetics.110.119867 ◽

2010 ◽

Vol 186 (2) ◽

pp. 669-676 ◽

Cited By ~ 16

Author(s):

Kyoichi Sawamura ◽

Kazunori Maehara ◽

Shotaro Mashino ◽

Tatsuo Kagesawa ◽

Miyuki Kajiwara ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Drosophila Simulans ◽

Female Sterility ◽

Nuclear Pore ◽

Nuclear Pore Protein ◽

Pore Protein

Download Full-text

Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.24.9595 ◽

1988 ◽

Vol 85 (24) ◽

pp. 9595-9599 ◽

Cited By ~ 43

Author(s):

M. D'Onofrio ◽

C. M. Starr ◽

M. K. Park ◽

G. D. Holt ◽

R. S. Haltiwanger ◽

...

Keyword(s):

Cdna Sequence ◽

Nuclear Pore ◽

Nuclear Pore Protein ◽

Partial Cdna Sequence ◽

Partial Cdna ◽

Sequence Encoding ◽

Pore Protein

Download Full-text

Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication.

The Journal of Cell Biology ◽

10.1083/jcb.128.5.721 ◽

1995 ◽

Vol 128 (5) ◽

pp. 721-736 ◽

Cited By ~ 97

Author(s):

M A Powers ◽

C Macaulay ◽

F R Masiarz ◽

D J Forbes

Keyword(s):

Protein Import ◽

Polyclonal Antiserum ◽

Peptide Sequence ◽

Nuclear Pore ◽

Chromosomal Dna ◽

Nuclear Pore Protein ◽

Egg Extracts ◽

Xenopus Egg ◽

Punctate Pattern

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.

Download Full-text