Identification of structural elements in Nox1 and Nox4 controlling localization and activity.
Nox NADPH oxidases differ in their mode of activation, subcellular localization, and physiological function. Nox1 releases superoxide anions (O(2)(-)) and depends on cytosolic activator proteins, whereas Nox4 extracellularly releases hydrogen peroxide (H(2)O(2)), and its activity does not require cotransfection of additional proteins. We constructed chimeric proteins consisting of Nox1 and Nox4 expressed in HEK293 cells. When the cytosolic tail of Nox4 was fused with the transmembrane part of Nox1, Nox1 became constitutively active. The reciprocal construct was inactive, suggesting that cytosolic subunit-dependent activation requires elements in the transmembrane loops. By TIRF-microscopy, Nox1 was observed in the plasma membrane, whereas Nox4 colocalized with proteins of the endoplasmic reticulum. Fusion proteins of Myc and Nox revealed that the N-terminal part of Nox1 but not Nox4 is cleaved. When the potential signal peptide of Nox4 was inserted into Nox1, plasma-membrane localization was lost, and the protein was retained in vesicle-like structures below the plasma membrane. The potential signal peptide of Nox1 failed to translocate Nox4 to the plasma membrane but switched the extracellularly detectable ROS from H(2)O(2) to O(2)(-). Thus, the very N-terminal part of Nox proteins determines subcellular localization and the ROS type released, whereas the cytosolic tail regulates activity.
Helmcke
Ina
I
Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Frankfurt am Main, Germany.
Heumüller
Sabine
S
Tikkanen
Ritva
R
Schröder
Katrin
K
Brandes
Ralf P
RP
D015153:Blotting, Western; D002460:Cell Line; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D021382:Protein Sorting Signals; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C498910:NOX4 protein, human