Novel properties of the nucleolar targeting signal of human angiogenin.
The polypeptide ligand angiogenin, a potent inducer of angiogenesis, localizes in the nucleus/nucleolus subsequent to endocytosis by relevant cell types. This study examines the kinetic properties of the nucleolar targeting signal (NTS) of angiogenin (IMRRRGL(35)) at the single cell level. We show that the NTS is sufficient to target green fluorescent protein (GFP), but not beta-galactosidase, to the nucleolus of rat hepatoma cells. Mutation of Arg(33) to Ala within the NTS abolishes targeting activity. Nuclear/nucleolar import conferred by the NTS of angiogenin is reduced by cytosolic factors as well as ATP and is independent of importins and Ran. The NTS also confers the ability to bind to nuclear/nucleolar components which is inhibited by ATP hydrolysis; nonhydrolysable GTP analogs prevent nuclear accumulation in the absence of an intact nuclear envelope through an apparent cytoplasmic retention mechanism. Since the lectin wheat germ agglutinin does not inhibit transport, we postulate a mechanism for angiogenin nuclear/nucleolar import involving passive diffusion of angiogenin through the nuclear pore and NTS-mediated nuclear/nucleolar retention, and with cytoplasmic retention modulating the process. This pathway is clearly distinct from that of conventional signal-mediated nuclear protein import.
Lixin
R
R
Nuclear Signalling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra City, Australia.
Efthymiadis
A
A
Henderson
B
B
Jans
D A
DA
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002793:Cholic Acids / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014909:Wheat Germ Agglutinins / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D002793:Cholic Acids; D003902:Detergents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014909:Wheat Germ Agglutinins; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; C046366:angiogenin; D012259:Ribonuclease, Pancreatic; D001616:beta-Galactosidase; D020931:ran GTP-Binding Protein; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate