Nuclear shuttling of the peptidase nardilysin.
The metalloendopeptidase nardilysin contains a putative N-terminal nuclear localization signal. The functionality of this sequence was tested with nardilysin-GFP fusion constructs. Expression in NIH3T3 cells showed approximately 90-95% of nardilysin-GFP as cytoplasmic. However, 3-6% of transfected cells showed both cytosolic and nuclear staining, while 2-4% showed predominantly nuclear staining. A nuclear localization signal mutant and an N-terminally truncated nardilysin-GFP with the nuclear localization signal deleted were completely cytoplasmic. Although endogenous nardilysin was barely detectable in the nucleus, after treatment with leptomycin B, nuclear nardilysin rose to approximately 15% and to over 25% after addition of spermine. The ability of a methionine 49 to act as the sole initiator methionine, as previously proposed, was tested by inserting a c-myc epitope between leucine28 and glycine29. Expression in HEK293 cells showed the presence of the c-myc tag, demonstrating that the enzyme can be translated from the first methionine and contains the nuclear localization signal.
Ma
Zhangliang
Z
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA.
Chow
K Martin
KM
Yao
Jia
J
Hersh
Louis B
LB
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D003600:Cytosol / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013096:Spermine / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D005231:Fatty Acids, Unsaturated; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013096:Spermine; D008666:Metalloendopeptidases; C087976:nardilysin; C038753:leptomycin B