Isolation and characterization of nuclear envelopes from the yeast Saccharomyces.
We have developed a large scale enrichment procedure to prepare yeast nuclear envelopes (NEs). These NEs can be stripped of peripheral proteins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents revealed previously uncharacterized ring structures associated with the NE that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedure allowed balance-sheet tabulation of the subcellular distribution of various NE and non-NE proteins. Thus we found that 20% of endoplasmic reticulum (ER) marker proteins are localized at the NE. Using a novel monospecific mAb made against proteins in the H-NE fraction and found to be directed against the pore membrane protein POM152, we showed that while the majority of POM152 is localized in the NE at the NPC, a proportion of this protein is also present in the ER. This ER pool of POM152 is likely to be involved in the duplication of nuclear pores and NPCs during S-phase. Both the NEs and H-NEs were found to be competent for the in vitro posttranslational translocation of prepro-alpha-factor. They may also be suitable to investigate other ER- and NE-associated functions in cell-free systems.
Strambio-de-Castillia
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Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York 10021, USA.
Blobel
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D000911:Antibodies, Monoclonal; D001692:Biological Transport; D015153:Blotting, Western; D002458:Cell Fractionation / Q000379:methods*; D002474:Cell-Free System / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D009685:Nuclear Envelope / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure*; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure*
D000911:Antibodies, Monoclonal; D003902:Detergents; D008565:Membrane Proteins; D006493:Heparin