Assembly/Disassembly of the Nuclear Envelope Membrane

doi:10.1074/jbc.270.32.19066

Assembly/Disassembly of the Nuclear Envelope Membrane

CHARACTERIZATION OF THE MEMBRANE-CHROMATIN INTERACTION USING PARTIALLY PURIFIED REGULATORY ENZYMES (*)

Rupert Pfaller and
John W. Newport(^§)

From theDepartment of Biology, University of California, San Diego, La Jolla, California 92093-0347

^§ To whom correspondence should be addressed:
Dept. of Biology, 0347, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0347.
Tel.: 619-534-3423; Fax: 619-534-0555.

Abstract

Assembly and disassembly of the nucleus at mitosis in eukaryotes involves the reversible interaction of chromatin with the nuclear membrane. Previously we have shown that this interaction is regulated by the antagonistic activities of a kinase and a phosphatase. The kinase promotes membrane release while the phosphatase stimulates binding. In this report we describe four steps in the purification of the kinase needed for release of membranes from chromatin. We also show that the release kinase and the mitotic initiation kinase, cdc2, are distinct and are separated from each other during the second purification step. Reconstitution experiments using these two kinases demonstrate that the release kinase and cdc2 kinase work in concert to cause membrane release from chromatin. In phosphorylation experiments, protein targets that are substrates for the regulatory release kinase are identified on the membranes. These phosphorylated proteins are candidates for regulated proteins mediating membrane-chromatin interaction. Finally, we find that membrane release activity can also be extracted from membranes by high salt treatment, indicating a possible dual localization of this activity.

Footnotes

↵* This work was supported by a fellowship from the Deutsche Forschungsgemeinschaft (to R. P.) and by National Institutes of Health Grant GM 33523 (to J. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ATPS

adenosine 5′-3-O-(thio)triphosphate

GTPS

guanosine 5′-3-O-(thio)triphosphate

DMAP

6,6′-dimethyl aminopurine.
↵²R. Pfaller and J. Newport, unpublished observations.
↵³R. Pfaller and J. Newport, unpublished results.
- Received May 5, 1995.
- Revision received June 6, 1995.