Signal- 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 into the nucleus through the nuclear pore complex by a mechanism that is not well understood. To better characterize the mechanisms of transport, we have established an homologous in vitro system using an NTS-containing structural protein of simian virus 40 (SV40) and isolated nuclei from cultured cells of its natural host. Isolated nuclei accumulated either fluorescently labeled SV40 Vp3-NTS peptide-BSA conjugates (NTSwt-BSA), as assayed cytochemically, or 125I-NTSwt-BSA, as assayed by filtration, in a signal- and ATP-dependent manner. Nuclear accumulation required nuclear membrane integrity and was inhibited by the lectin wheat germ agglutinin but not concanavalin A. Unlike several other systems, this system is not dependent on cytoplasmic extracts for the transport of SV40 proteins. NTSwt-BSA was transported with an apparent Km of 0.8 microM and Vmax of 0.8 nmol/min/10(*) nuclei. Thin section autoradiography confirmed the transport. This system faithfully reproduced what occurs in vivo: nuclear import of the SV40 capsid protein Vp3 was dependent on the presence of its functional NTS. Full-length Vp3, expressed 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.
Dean
D A
DA
Department of Biology, University of California, Los Angeles 90024.
Kasamatsu
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002213:Capsid / Q000096:biosynthesis / Q000378:metabolism*; D036022:Capsid Proteins; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002999:Clone Cells; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D005982:Glutathione Transferase / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009865:Oocytes / Q000502:physiology; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013539:Simian virus 40 / Q000378:metabolism*; D014020:Tissue Extracts; D014764:Viral Proteins / Q000378:metabolism*; D014981:Xenopus
D036022:Capsid Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014020:Tissue Extracts; C085902:VP3 protein, Simian virus 40; D014764:Viral Proteins; D005982:Glutathione Transferase