Multiple nuclear localization sequences allow modulation of 5-lipoxygenase nuclear import.
The nuclear import of proteins typically requires the presence of a nuclear localization sequence (NLS). Some proteins have more than one NLS, but the significance of having multiple NLSs is unclear. The enzyme 5-lipoxygenase (5-LO) has three NLSs that, unlike the tight cluster of basic residues of the classical SV40 large T antigen NLS, contain dispersed basic residues. When attached to green fluorescent protein (GFP), individual 5-LO NLSs caused quantitatively and statistically less import than the SV40 NLS. Combined 5-LO NLSs produced nuclear import that was comparable to that of the SV40 NLS. As expected, GFP/NLS proteins displayed relatively uniform import in all cells. However, a fusion protein of GFP plus the 5-LO protein, modified to contain only one functional NLS, produced some cells with import and some cells without import. A GFP/5-LO fusion protein containing two functional NLSs produced four identifiable levels of nuclear import. Quantitative and visual analysis of a population of cells expressing the intact GFP/5-LO protein, with three intact NLSs, indicated five levels of nuclear import. This suggested that the subcellular distribution of 5-LO may vary widely in normal cells of the body. Consistent with this, immunohistochemical staining of lung sections found that individual macrophages, in situ, displayed cell-specific levels of import of 5-LO. Since nuclear accumulation is known to affect 5-LO activity, multiple NLSs may allow graded regulation of activity via controlled import. Multiple NLSs on other proteins may likewise allow fine control of protein action through modulation of the level of import.
Department of Internal Medicine, University of Michigan, 6301 MSRB III, Ann Arbor, MI 48109, USA.
Ming
M
Luo
C W Mark
CW
Pang
Alison E
AE
Gerken
Thomas G
TG
Brock
D021581:Active Transport, Cell Nucleus; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D008168:Lung / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals; D010957:Plasmids / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001094:Arachidonate 5-Lipoxygenase