Colocalization of vertebrate lamin B and lamin B receptor (LBR) in nuclear envelopes and in LBR-induced membrane stacks of the yeast Saccharomyces cerevisiae.
We have expressed human lamin B and the chicken lamin B receptor (LBR), either separately or together, in yeast and have monitored the subcellular location of the expressed proteins by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation. At the light microscopic level, the heterologous lamin B localized to the yeast nuclear rim and at electron microscopic resolution was found subjacent to the yeast inner nuclear membrane. These data indicate that vertebrate lamin B was correctly targeted in yeast. Expression of the heterologous LBR, either alone or together with the heterologous lamin B, resulted in the formation of membrane stacks primarily adjacent to the nuclear envelope, but also projecting from the nuclear envelope into the cytoplasm or under the plasma membrane. Double immunoelectron microscopy showed colocalization of the heterologous lamin B and LBR in the yeast nuclear envelope and in the LBR-induced membrane stacks. Cell fractionation showed the presence of the heterologous lamin B and LBR in a subnuclear fraction enriched in nuclear envelopes. The heterologous lamin B was extracted at 8 M urea, but not at 4 M urea, thus behaving as a peripheral membrane protein and indistinguishable from assembled lamins. The heterologous LBR was not extracted by 8 M urea, indicating that it was integrated into the membrane. The observed colocalization and cofractionation are consistent with previously reported in vitro binding data and suggest that heterologous lamin B and LBR interact with each other when coexpressed in yeast.
Smith
S
S
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, NY 10021.
Blobel
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G
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D034921:Lamin Type B; D034882:Lamins; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D009685:Nuclear Envelope / Q000378:metabolism* / Q000648:ultrastructure; D009687:Nuclear Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D000097682:Lamin B Receptor
D018076:DNA, Complementary; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D000097682:Lamin B Receptor