SIGNAL-DEPENDENT AND ENERGY-DEPENDENT NUCLEAR TRANSPORT OF SV40 VP3 BY ISOLATED-NUCLEI - ESTABLISHMENT OF A FILTRATION ASSAY FOR NUCLEAR-PROTEIN IMPORT

Nuclear transport signal (NTS)-containing proteins are transported int o the nucleus through the nuclear pore complex by a mechanism that is not well understood. To better characterize the mechanisms of transpor t, we have established an homologous in vitro system using an NTS-cont aining structural protein of simian virus 40 (SV40) and isolated nucle i from cultured cells of its natural host. Isolated nuclei accumulated either fluorescently labeled SV40 Vp3-NTS peptide-BSA conjugates (NTS wt-BSA), as assayed cytochemically, or I-125-NTSwt-BSA, as assayed by filtration, in a signal- and ATP-dependent manner. Nuclear accumulatio n required nuclear membrane integrity and was inhibited by the lectin wheat germ agglutinin but not concanavalin A. Unlike several other sys tems, this system is not dependent on cytoplasmic extracts for the tra nsport of SV40 proteins. NTSwt-BSA was transported with an apparent K- m of 0.8 mu m and V-max of 0.8 nmol/min/10(8) nuclei. Thin section aut oradiography confirmed the transport. This system faithfully reproduce d what occurs in vivo: nuclear import of the SV40 capsid protein Vp3 w as dependent on the presence of its functional NTS. Full-length Vp3, e xpressed as a glutathione S-transferase fusion protein, and a deletion mutant which retains its NTS, Vp3 Delta C13, were transported by the nuclei but Vp3 Delta C35, which lacks the NTS, and an NTS mutant, VP3( 202E/204T), were not transported.