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 prote ins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents rev ealed previously uncharacterized ring structures associated with the N E that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedu re 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 th at while the majority of POM152 is localized in the NE at the NPC, a p roportion of this protein is also present in the ER. This ER pool of P OM152 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 competen t for the in vitro posttranslational translocation of prepro-alpha-fac tor. They may also be suitable to investigate other ER- and NE-associa ted functions in cell-free systems.