Translocation of NLS–BSA conjugates into nuclei of permeabilized mammalian cells can be supported by protoplast extract: An experimental system for studying plant cytosolic factors involved in nuclear import

An heterologous experimental system, which allows the study of the yet unknown cytosolic factors involved in nuclear import of nuclear localization signal (NLS)-containing proteins in plants, has been established. The ability of plant cell extract to substitute mammalian cytosol and to promote translocation of NLS-containing proteins into nuclei of permeabilized HeLa cells was demonstrated. The results described in the present work show that nuclear import of fluorescently labeled BSA conjugates bearing the NLS sequence of SV40 large T antigen could be supported by petunia cell cytoplasmic extract. This heterologous system shows the characteristic features of the homologous mammalian system, namely, is ATP dependent and is inhibited by WGA, GTPγS as well as by non-fluorescent NLS–BSA conjugates. The system described here offers an experimental method to study and characterise cytosolic factors which are required for nuclear import in plants.