In vitro translocation through the yeast nuclear envelope. Signal-dependent transport requires ATP and calcium

Abstract

Nuclei rapidly purified from yeast Saccharomyces cerevisiae using a cytochalasin B enucleation procedure are substantially free of cell wall, secretory vesicle, plasma membrane vacuolar, and cytoplasmic and mitochondrial contamination. Nuclei obtained in this manner in high yield retain transport properties comparable to nuclei in situ. An in vitro nuclear import assay system has been developed using isolated nuclei and radiolabeled proteins prepared by a coupled in vitro transcription/translation system. Both wild-type SV40 large T-antigen and nucleoplasmin are imported into isolated yeast nuclei, whereas a missense cytoplasmic mutant of the SV40 large T-antigen (Lys128----Thr) and cytoplasmic dihydrofolate reductase are not imported. Association and import of these proteins in a time- and signal-dependent manner resulted in their protection from trypsin that was tethered to agarose beads. Greater than 70% of the labeled protein harboring a karyophilic signal was imported in a reaction that could be blocked by prior treatment of nuclei with trypsin-agarose. Nuclear accumulation of SV40 large T-antigen and nucleoplasmin was unidirectional, ATP and Ca2+ dependent, and was not inhibited by a vast excess of exogenous nonnuclear competitor protein. This system provides an important new tool in combination with powerful yeast genetic methods for analysis of the mechanism and the apparatus for transport at the nuclear envelope.