Subcellular distribution of the Xenopus p58/lamin B receptor in oocytes and eggs.
The p58/lamin B receptor of vertebrates is localized in the inner nuclear membrane. Antibodies raised against the bacterially expressed amino-terminal half of Xenopus p58 (Xp58) revealed that in Xenopus oocytes the vast majority of this membrane protein is localized in cytoplasmic membranes. Only very small amounts of p58 not detectable by immunofluorescence microscopy were contained in the oocyte nuclear envelope. In contrast, nuclear membranes of 2-cell stage embryos were successfully stained with p58 antibodies, nuclei reconstituted in vitro in Xenopus egg extracts contained p58, and the nucleoplasmic domain of Xp58 could be specifically bound to sperm chromatin in vitro. One major difference between oocytes and early embryonic cells is that no chromatin is associated with the oocyte inner nuclear membrane whereas the complement of lamins is identical in both cell types. To gain insight into the properties of oocyte p58 we microinjected isolated nuclei of cultured rat cells into the cytoplasm of Xenopus oocytes. The oocyte p58 was detectable by immunofluorescence microscopy within 16-20 hours in the nuclear membrane of rat nuclei. Our data indicate that the peripheral chromatin but not lamins are required for the retention of p58 in the inner nuclear membrane. Sucrose step gradient centrifugation of total oocyte membranes revealed that the oocyte p58 was predominantly recovered in membrane fractions that did not contain lamins whereas membrane associated lamins and p58 of unfertilized eggs were found in the same fractions. By electron microscopical immunolocalizations one major population of meiotic p58 vesicles was identified that contained exclusively p58 and a second minor population (ca. 11% of p58 vesicles) contained in addition to p58 membrane bound B-type lamins. Egg vesicles containing pore membrane proteins were predominantly recovered in gradient fractions that did not contain p58 and B-type lamins. Our data indicate that the targeting of p58 to chromatin at the end of mitosis in the early Xenopus embryo is a process independent from that of lamin targeting. Comparable to the situation in oocytes and eggs, a significant proportion of p58 of interphase cells could be recovered in fractions that did not contain lamins. This population of p58 molecules could be extracted from A6-cells with buffers containing 1% Triton X-100/0.15 M NaCl and could be pelleted by a 50,000 g centrifugation. A- and B-type lamins were not detectable in the p58 containing pellet.
Gajewski
A
A
Division of Electron Microscopy, Biocenter of the University of Würzburg, Am Hubland, D-97074 Würzburg, Germany.
Krohne
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D002645:Chickens; D002843:Chromatin / Q000502:physiology / Q000648:ultrastructure*; D003594:Cytoplasmic Granules / Q000502:physiology / Q000648:ultrastructure; D004625:Embryo, Nonmammalian / Q000502:physiology / Q000648:ultrastructure; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D008540:Meiosis; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000502:physiology / Q000648:ultrastructure; D009865:Oocytes / Q000502:physiology* / Q000648:ultrastructure; D010063:Ovum / Q000502:physiology* / Q000648:ultrastructure; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013094:Spermatozoa / Q000502:physiology* / Q000648:ultrastructure; D014982:Xenopus laevis; D000097682:Lamin B Receptor
D002843:Chromatin; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D000097682:Lamin B Receptor