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 m embrane. Antibodies raised against the bacterially expressed amino-terminal half of Xenopus p58 (Xp58) revealed that in Xenopus oocytes the vast major ity of this membrane protein is localized in cytoplasmic membranes. Only ve ry small amounts of p58 not detectable by immunofluorescence microscopy wer e contained in the oocyte nuclear envelope. In contrast, nuclear membranes of 2-cell stage embryos were successfully stained with p58 antibodies, nucl ei reconstituted in vitro in Xenopus egg extracts contained p58, and the nu cleoplasmic domain of Xp58 could be specifically bound to sperm chromatin i n vitro. One major difference between oocytes and early embryonic cells is that no chromatin is associated with the oocyte inner nuclear membrane wher eas the complement of lamins is identical in both cell types. To gain insig ht into the properties of oocyte p58 we microinjected isolated nuclei of cu ltured rat cells into the cytoplasm of Xenopus oocytes, The oocyte p58 was detectable by immunofluorescence microscopy within 16-20 hours in the nucle ar 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 m embrane. Sucrose step gradient centrifugation of total oocyte membranes revealed tha t the oocyte p58 was predominantly recovered in membrane fractions that did not contain lamins whereas membrane associated lamins and p58 of unfertili zed eggs were found in the same fractions, By electron microscopical immuno localizations one major population of meiotic p58 vesicles was identified t hat contained exclusively p58 and a second minor population (ca, 11% of p58 vesicles) contained in addition to p58 membrane bound B-type lamins, Egg v esicles containing pore membrane proteins were predominantly recovered in g radient fractions that did not contain p58 and B-type lamins. Our data indi cate that the targeting of p58 to chromatin at the end of mitosis in the ea rly Xenopus embryo is a process independent from that of lamin targeting. Comparable to the situation in oocytes and eggs, a significant proportion o f p58 of interphase cells could be recovered in fractions that did not cont ain lamins, This population of p58 molecules could be extracted from A6-cel ls with buffers containing 1% Triton X-100/0,15 M NaCl and could be pellete d by a 50,000 g centrifuation, A- and B-type lamins were not detectable in the p58 containing pellet.