Subcellular localization and function of alternatively spliced Noxo1 isoforms

Abstract

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. Noxo1alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1alpha and delta 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 Noxo1beta supports the highest activity and Noxo1gamma and Noxo1alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma 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.

Fig. 1. Alternative splicing of Noxo1 transcripts and its affects on PX domain structure

A, Schematic of Noxo1 gene organization, showing locations of alternative splice sites on both boundaries of exon 3 (blue and red). Exons are shown as numbered boxes, while introns are represented as thin lines. B, Alignment of human and mouse Noxo1 genomic sequences around exon 3. Capital and bold letters show exonic sequences, while small letters denote intronic sequences. Amino acids encoded by alternative splicing are shown in blue and red at the beginning and end of exon 3, respectively. Arrows and arrowheads indicate alternative splice donor and splice acceptor sites, respectively. C, Alignment of Noxo1 isoform PX domain sequences with those of other PX domains (conserved amino acids shown in pink). Conserved secondary structural elements identified within the three-dimensional structure of p47^phox are shown above sequences (α, alpha helix; β, beta sheet)[34,36]. Amino acids resulting from alternative splicing at sites 1 and 2 are shown in blue and red, respectively. Splice site 1 (1) occurs in the middle of conserved α-helix 1, while splice site 2 (2) occurs within a variable sequence that includes a proline-rich motif that interacts with SH3 domains in some proteins. Contact residues within the phosphatidylinositol-binding pocket of p47^phox are overlined, while those lining a second phosphatidic acid (PA) binding pocket are marked with an asterisk. GeneBank™ accession no. of Noxo1α, Noxo1β, Noxo1γ, Noxo1δ, p47^phox, p40^phox, PLD1 are NM_144603, NM_172167, NM_172168, AY191359, NM_000265, NM_000631, NM_002662, respectively. FASTA™ accession no. of Bem1 and Vam7p are NP_009759 and NP_011303, respectively.

Fig. 2. Subcellular localization of Noxo1(PX)-GFP and Noxo1-GFP in two tranfected cell models

A, Noxo1α(PX)-GFP and Noxo1α-GFP are localized on intracellular vesicles or in large cytoplasmic aggregates, but not on the plasma membrane in HEK293 cells. Nuclei are stained (blue) using Hoechst 33258. Noxo1β(PX)-GFP and Noxo1β-GFP are localized predominately along the plasma membrane in HEK293 cells. Noxo1γ(PX)-GFP and Noxo1γ-GFP localize in the nucleus and weakly along the plasma membrane, particularly Noxo1γ(PX)-GFP. Noxo1δ(PX)-GFP and Noxo1δ-GFP show localization patterns similar to that of Noxo1α, and no nuclear localization, as observed with Noxo1γ(PX)-GFP and Noxo1γ-GFP. Bar: 10 μm B, Noxo1α(PX)-GFP and Noxo1α-GFP are detected at low levels on the plasma membrane (arrows), as well as on intracellular vesicles of COS-7 cells. Noxo1β(PX)-GFP and Noxo1β-GFP are localized on the plasma membrane. Noxo1γ(PX)-GFP and Noxo1γ-GFP are again localized on the plasma membrane and in the nucleus of COS7 cells. Noxo1δ(PX)-GFP and Noxo1δ-GFP show localization patterns similar to Noxo1α and no significant nuclear association. Plasma membrane localization of Noxo1δ(PX)-GFP and Noxo1δ-GFP is less than that seen with Noxo1α(PX)-GFP and Noxo1α-GFP. C, The conserved Arg 40 mutation apparently disrupts the association of Noxo1β-GFP with the plasma membrane (arrow) in HEK293 cells. Bar: 10 μm D, Western blotting confirms comparable levels and stability of all four Noxo1 fusion proteins in HEK293 and COS-7 cells. Comparable sample loading is confirmed by β-tubulin immunoblotting. Results are representative of those obtained in three separate experiments

Fig. 3. Activation of Nox1 by Noxo1α, Noxo1β, and Noxo1γ in three transfected cell models

In HEK293 (A) and in CHO-K1 (C) cells, Nox1 activity is efficiently supported by Noxo1β, moderately by Noxo1γ, and poorly by Noxo1α. In contrast, in COS-7 cells (C) Nox1 activity is supported at similar levels by all three Noxo1 isoforms. The enhancing effects of PMA are greater in HEK293 and CHO-K1 cell models than the COS-7 cell model. Dominant negative effects of p22^phox(P156Q) are seen with all three isoforms in all cell models, but is most apparent with Noxo1α in all cell models. D, Nox1 activity reconstituted by Noxo1β in the CHO cell model is a fraction of that observed in the other two models. Data are from at least three independent experiments

Fig. 4. Phospholipid binding of PX domain of Noxo1 isoforms

A, SDS-PAGE analysis of GST-Noxo1α reveals poor yield of the intact 43 kDa fusion protein and a major GST degradation product (25kDa). Intact GST-Noxo1β(PX) and GST-Noxo1γ(PX) are detected at expected molecular weights (~43 kDa). B, GST-Noxo1β binds to PtdIns-(3)P, PtdIns-(4)P, and PtdIns-(5)P, PtdIns-(3,5)P₂, and phosphatidic acid (PA). GST-Noxo1γ shows almost the same binding pattern, while GST shows no lipid binding. LPA, Lysophosphatidic acid; LPC, Lysophosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylcholine; S1P, sphingosine 1-phosphate; PS, phosphatidylserine Results shown were similar in three independent experiments.

Fig. 5. Tissue-specific expression patterns of Noxo1 isoform mRNAs

A, Noxo1 mRNAs (arrow) are detected by RT-PCR using single-strand cDNA templates prepared from colon, testis (test), liver, and pancreas (panc). Noxo1 mRNA is faintly detected in pancreas. M, marker; cont, control reaction without cDNA template. Noxo1β and Noxo1γ cDNAs are detected from all tissues, however, Noxo1α is detected in testis and liver, and Noxo1δ is detected only in liver. C, Alternatively-spliced Noxo1-GFP isoforms show distinct subcellular distribution patterns in pancreatic (PANC-1) and testis (TM3) epithelial cell lines. Noxo1α is predominately vesicular (arrows: the plasma membrane), Noxo1β associates with the plasma membrane, and Noxo1γ is both in nuclei and on the plasma membrane. Bar: 10 μm.

Fig. 6. Nox1-dependent, but Noxo1-independent, targeting of endogenous p22^phox to the plasma membrane

A, Immunofluorescence imaging of endogenous and transfected p22^phox in HEK293 cells, showing a reticular cytoplasmic and peri-nuclear membrane (arrows) staining patterns. Transfection of Nox1 results in a redistribution of endogenous p22^phox to the plasma membrane. *, control cells not transfected with Nox1 show much weaker staining of endogenous p22^phox, primarily in a reticular cytoplasmic pattern. B, Transfection of Noxo1(α, β, or γ)-GFP shows no redistribution of endogenous p22^phox. C, Western blotting shows increased p22^phox levels in Nox1-transfected, but not in mock or Noxo1β-transfected cells. Comparable protein loading is confirmed by β-tubulin immunoblotting. Similar results were obtained in three independent experiments.