Subcellular localization and function of alternatively spliced Noxo1 isoforms

• Published in: Free radical biology & medicine Vol. 42; no. 2; pp. 180 - 190
• Main Authors: Ueyama, Takehiko, Lekstrom, Kristen, Tsujibe, Satoshi, Saito, Naoaki, Leto, Thomas L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.01.2007
• Subjects: Adaptor Proteins, Vesicular Transport - genetics; Adaptor Proteins, Vesicular Transport - metabolism; Adaptor Proteins, Vesicular Transport - ultrastructure; Alternative Splicing; Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cell Membrane - enzymology; Enzyme Activation - physiology; Humans; Immunoblotting; Isoenzymes - genetics; Isoenzymes - metabolism; Isoenzymes - ultrastructure; Microscopy, Confocal; Molecular Sequence Data; NADPH Oxidases - genetics; NADPH Oxidases - metabolism; NADPH Oxidases - ultrastructure; Reverse Transcriptase Polymerase Chain Reaction; Subcellular Fractions - enzymology; Subcellular Fractions - ultrastructure; Transfection; Ab; Noxo1; GST; ER; CHAPS; GFP; PA; SDS-PAGE; FBS; ROS; Noxa1; RFP; PCR
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2006.08.024

Nox organizer 1 (Noxo1), a p47 phox homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1α, β, γ, and δ show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1β exhibits prominent plasma membrane binding, Noxo1γ shows plasma membrane and nuclear associations, and Noxo1α and δ localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1β supports the highest activity and Noxo1γ and Noxo1α support moderate or low activities, respectively. In COS-7 cells, where Noxo1α localizes on the plasma membrane, the activities supported by the three isoforms (α, β, and γ) do not differ significantly. The PX domains of β and γ bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22 phox localization, although Nox1 is needed to transport p22 phox to the plasma membrane.