Human aquaporin-11 guarantees efficient transport of H2O2 across the endoplasmic reticulum membrane

Abstract

Hydrogen peroxide (H₂O₂) is an essential second intracellular messenger. To reach its targets in the cytosol, H₂O₂ must cross a membrane, a feat that requires aquaporins (AQP) endowed with 'peroxiporin' activity (AQP3, AQP8, AQP9). Here, we exploit different organelle-targeted H₂O₂-sensitive probes to show that also AQP11 efficiently conduits H₂O₂. Unlike other peroxiporins, AQP11 is localized in the endoplasmic reticulum (ER), accumulating partly in mitochondrial-associated ER membranes (MAM). Its downregulation severely perturbs the flux of H₂O₂ through the ER, but not through the mitochondrial or plasma membranes. These properties make AQP11 a potential regulator of ER redox homeostasis and signaling.

Keywords: Aquaporins; Endoplasmic reticulum; Hydrogen peroxide; Membrane permeability; Peroxiporins; Redox homeostasis.

Graphical abstract

Fig. 1

AQP11 resides in the ER. A. HeLa transfectants expressing HaloAQP11 were co-stained with fluorescent Halo ligands, and with antibodies against calnexin (CNX) or peroxiredoxin 3 (PRX3), to decorate AQP11, the ER and mitochondria, respectively. To label glycoproteins on the plasma membrane, cells were stained with concanavalin A-FITC (ConA) in absence of cell permeabilization, and then fixed (middle panels). Scale bar = 10 μm. B. HeLa cells expressing AQP11mycFlag were fractionated as previously described [22,23]. Aliquots were resolved electrophoretically and stripes of the blots decorated with the indicated antibodies. Hom, total post-nuclear homogenates. Cyt, cytosol. MP, pure mitochondrial fraction. MAM, mitochondria-associated membranes. ER, endoplasmic reticulum.

Fig. 2

Both the N- and C- termini of AQP11 protrude into the cytosol. A. HeLa transfectants expressing HaloAQP11-Flag were treated with or without digitonin to selectively solubilize the plasma membrane, and then incubated with a membrane-impermeant Halo ligand (green) or with antibodies specific for Flag (cyan) or calreticulin (CRT, red), a lumenal protein of the ER. See Supplementary Fig. S2 for further details on the protocol. Note that both the N- and C-termini of HaloAQP11-Flag face the cytosol, as schematized in panel B (top scheme). Scale bar = 10 μm. B. The top panel shows the topology of AQP11 and AQP8 (orange and blue, respectively) as determined by immunofluorescence (Fig. 2A) and biochemical analyses (bottom panel). The absence of N-glycosylated AQP11 species in HeLa transfectants contrasts with the easily detected Endo-H resistant AQP8 glycoforms. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

AQP11 forms tetramers. A. Aliquots of the lysates of HeLa co-transfectants expressing AQP11mycFlag and either Halo-RDEL, HaloGpx8 TM or HaloAQP11 proteins were immunoprecipitated using immobilized anti-Flag antibodies and analyzed by western blotting using anti-Halo or anti-Flag specific antibodies. IP, immunoprecipitation. B. Lysates of HeLa cells expressing either HaloAQP11-Flag or HaloAQP8-Flag were separated through a discontinuous 5– 11% sucrose density gradient, and aliquots of each fraction subjected to reducing SDS-PAGE. The presence of the recombinant proteins was assessed by direct detection of bound fluorescent Halo ligands on the gels using a laser scanner imager.

Fig. 4

AQP11 is a peroxiporin. A. Polyclonal HeLa cells stably expressing HyPer in the ER lumen (HyPerERLum) or in the cytosol (HyPerCyto) were treated with AQP11-specific or control small interfering RNAs (siRNAs) and the kinetics of probe activation upon exposure to 50 μM H₂O₂ plotted against time. The black arrow indicates when H₂O₂ was added. Data are shown as mean fold changes of the 488/405 nm ratio and corresponds to ≥5 experiments ± standard error of the mean (SEM). Note that AQP11 silencing inhibits HyPerERLum activation without interfering with the cytosolic sensor. B. Measurement of the basal oxidation state of HyPerERLum and HyPerCyto. Each dot represents a single cell distributed in the graph according to its 488/405 nm ratio. Comparing the panels highlights that AQP11 silencing impacts in opposite ways the ER lumenal and cytosolic HyPer sensors.

Fig. 5

AQP11 restores H₂O₂fluxes in silenced cells.HyPerERLum HeLa transfectants were silenced for AQP11 expression and then transfected with a silencing-resistant HaloAQP11. A. Representative immunofluorescence analyses of one of the experiments averaged in panel B confirm the lower response to exogenous H₂O₂ (top panels, HyPerERLum fold changes, in orange) and higher basal oxidation of HyPer ER lumen (middle panels, basal ratio, in rainbow) in AQP11-silenced cells. Both features are rescued by re-expression of HaloAQP11, as monitored using fluorescent Halo ligands as shown in the lower panel (in white). B. The efficiency of the entry of exogenous H₂O₂ (50 μM, top graph) and the basal oxidation state of the HyPer ER lumenal probe (bottom panel) were measured as in Fig. 4, in control or AQP11_low cells, before or after rescuing. Results are represented as percentage of H₂O₂ transported 1.5 min after of H₂O₂ addition relative to controls (Mock), and as the fold change in the basal 488/405 nm ratio (bottom). Average on ≥3 experiments ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)