Characterization of the binding of nuclear envelope precursor vesicles and chromatin, and purification of the vesicles.
The binding of nuclear envelope precursor vesicles and chromatin was characterized by using an in vitro system constituted from a Xenopus egg extract and demembranated Xenopus sperm chromatin. The results of binding studies in the presence of salts, urea, and a chelator showed that the binding involves an ionic interaction. Chemical modification studies suggested that a protein(s) in the vesicles, which is responsible for the binding with chromatin, has essential lysine, histidine, and methionine residues. The vesicle protein could not be extracted from vesicles with 1 M KCl, 2 M urea, or 0.1 M Na2CO3, suggesting that it is an intrinsic membrane protein. The protein was denatured with 8 M urea and 0.1 M Na2CO3, and could be renatured by incubation at 23 degrees C, suggesting that the native conformation of the protein is important for the binding. Affinity purification of nuclear envelope precursor vesicles was achieved by binding to chromatin and dissociation with 0.24 M NaCl. The vesicle fraction thus obtained exhibited the ability to form nuclear envelope on incubation with chromatin in Xenopus egg cytosol without any other membrane fraction. These results suggested that there is a nuclear envelope precursor vesicle population containing both a chromatin targeting protein and vesicle fusion machinery.
Imai
N
N
Department of Biochemistry, Faculty of Science, Niigata University.
Sasagawa
S
S
Yamamoto
A
A
Kikuchi
F
F
Sekiya
K
K
Ichimura
T
T
Omata
S
S
Horigome
T
T
D000818:Animals; D002498:Centrifugation; D011232:Chemical Precipitation; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D007096:Imidoesters / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009685:Nuclear Envelope / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013094:Spermatozoa; D014357:Trypsin / Q000494:pharmacology; D014981:Xenopus
D002843:Chromatin; D007096:Imidoesters; D008565:Membrane Proteins; D011498:Protein Precursors; C029326:dimethyl pimelimidate; D014357:Trypsin