Abstract
During each cell cycle, the nucleus of higher eukaryotes undergoes a dramatic assembly and disassembly. These events can be faithfully reproduced in vitro using cell-free extracts derived from Xenopus eggs. Such extracts contain three major N-acetylglucosaminylated proteins, p200, p97, and p60. All three become assembled into reconstituted nuclear pores. Here we show that p200, p97, and p60 exist in eggs in soluble high molecular mass complexes of 1000, 450, and 600 kDA, respectively. The bulk of p60 is stably associated with proteins of 58 and 54 kDa, while p200 is associated with a fraction of p60 in a separate complex lacking p58 and p54. Upon examining the behavior of these proteins in the cell cycle, we find that p200 and p97 are highly phosphorylated at mitosis, both in vivo and in vitro. Moreover, in extracts that cycle between interphase and mitosis, p200 and p97 are specifically phosphorylated at mitosis. Corresponding with their mitotic phosphorylation, both p200 and p97 are specific substrates for purified mitotic Cdc2 kinase, whereas nucleoporin p60 is not. Analysis indicates that the size of the complexes containing the pore N-acetylglucosamine glycoproteins does not change during mitosis, suggesting that such complexes represent stable multicomponent modules into which the nucleus disassembles at mitosis.
Highlights
During each cell cycle, the nucleus of higher eukaryotes undergoes a dramatic assembly and disassembly
The mitotic reorganization of the eukaryotic cell architecture ensures the correct partitioning of the cellular contents to each of the daughter cells, These mitotic changes are seen most vividly in the dramatic alterations of the cell nucleus where the chromosomes condense, the mitotic spindle assembles, and the nuclear envelope breaks down [1,2,3], There is currently a great deal of evidence to indicate that movement of the cell cycle into mitosis is regulated by the Cdc2 protein kinase [4]
The lamin proteins form homo- and heteropolymers through strong coiled/coil interactions both in vivo and in vitro [23, 24]. Despite these strong interactions the lamina is a very dynamic network that both completely disassembles during mitosis and incorporates new lamin proteins during nuclear growth in interphase [20, 25], Lamins have been shown to be phosphorylated during interphase and hyperphosphorylated during mitosis [21], lamin-associated proteins of the inner nuclear membrane are phosphorylated during interphase and mitosis [26,27,28,29]
Summary
(Received for publication, August 19, 1994, and in revised form, October 17, 1994). From the Department of Biology, University of California at San Diego, La Jolla, California 92093-0347. A number of proteins that bind to and modulate microtubule dynamics have been isolated, and some of these proteins (MAP4, p220, and p47) are phosphorylated in a cell cycle-dependent fashion [13, 15,16,17,18] Another key mitotic reorganization event seen in higher eukaryotes is the disassembly of the nuclear envelope. In previous studies we used an in vitro nuclear reconstitution system to identify and characterize a family of nuclear pore glycoproteins required for nuclear transport [38, 39] In this reconstitution system, nuclei assemble when sperm chromatin is mixed with the cytosolic and membrane fractions of a. In this study we have used interphase and mitotic egg extracts in a first step to investigate whether known nuclear pore proteins are phosphorylated in a cell cycle-dependent manner. The distinct complexes that contain each of these glycoproteins will be discussed
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
