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A Rhesus channel in the coral symbiosome membrane suggests a novel mechanism to regulate NH3 and CO2 delivery to algal symbionts

Reef-building corals maintain an intracellular photosymbiotic association with dinoflagellate algae. As the algae are hosted inside the symbiosome, all metabolic exchanges must take place across the symbiosome membrane. Using functional studies in Xenopus oocytes, immunolocalization, and confocal Airyscan microscopy, we established that Acropora yongei Rh (ayRhp1) facilitates transmembrane NH3 and CO2 diffusion and that it is present in the symbiosome membrane. Furthermore, ayRhp1 abundance in the symbiosome membrane was highest around midday and lowest around midnight. We conclude that ayRhp1 mediates a symbiosomal NH4+-trapping mechanism that promotes nitrogen delivery to algae during the day—necessary to sustain photosynthesis—and restricts nitrogen delivery at night—to keep algae under nitrogen limitation. The role of ayRhp1-facilitated CO2 diffusion is less clear, but it may have implications for metabolic dysregulation between symbiotic partners and bleaching. This previously unknown mechanism expands our understanding of symbioses at the immediate animal-microbe interface, the symbiosome.

protein concentrations were determined using a Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). 4x Laemmli buffer (Bio-Rad) and 10% β-mercaptoethanol were added to samples before heating at 90°C for 3 min. 22.5 µg protein and 4 µl of Precision Plus Dual Color Protein Standards (Bio-Rad) were loaded into a 10% polyacrylamide SDS-PAGE gel in a Mini-Trans Blot Cell (Bio-Rad) with running buffer (25 mM Tris, 190 mM glycine, 0.1% SDS). Electrophoresis was run for 100 min at 100 V (4°C).
Following electrophoresis, the gel was washed in distilled water for 5 min and equilibrated in Towbin buffer (25 mM Tris pH 8.3, 192 mM glycine, 20% (v/v) methanol) for 15 min. Proteins were transferred from the gel onto a PVDF membrane using a Mini Tans-Blot Cell (Bio-Rad) overnight in Towbin buffer (0.09 A, 4°C). PVDF membranes were washed in Trisbuffered Saline + 0.1% Tween detergent (TBS-T) for 15 min on a shaker at room temperature to remove excess transfer buffer prior to blocking. Membranes were then blocked with 5% powdered fat-free milk in TBS-T for 1 h on a shaker at room temperature.
Following blocking, membranes were incubated overnight on a shaker (4°C) with anti-ayRhp1 primary antibody (0.216 μg/ml), primary antibody with 400x excess peptide on a molar base ('pre-absorption control'), or pre-immune serum (0.216 μg/ml) diluted in blocking buffer. Membranes were then washed with 4 x 15-min TBS-T washes prior to incubation with secondary antibody (goat anti-rabbit-HRP diluted 1:10,000, Bio-Rad) for 1 h on a shaker at room temperature. Membranes were again washed with 4 x 15-min TBS-T washes and a final 15-min TBS wash prior to band development with an ECL Prime Western Blot Detection Kit (GE Healthcare, Chicago, IL, USA) and imaged using a Chemidoc Imaging system (Bio-Rad).

Confirmation of ayRhp1 Protein Expression in Xenopus Oocytes
Using methods adapted from (83), ayRhp1-expressing and control Xenopus oocytes were added to ice-cold homogenization buffer (250 mM sucrose, 1 mM EDTA, 30 mM TRIS in milliQ-water) spiked with protease inhibitor cocktail (MilliporeSigma, Burlington, MA, USA) at a ratio of 1 oocyte:50 μL. Oocytes were homogenized on ice by gentle pipetting and left for 20 mins at 4°C to solubilize membrane proteins. Homogenates were centrifuged (10,000 x g, 2 min, 4°C) to pellet yolk and debris; the lipid layers on top of the supernatants were removed and discarded. The supernatants were transferred to new tubes on ice and saved as crude homogenates. Sample protein concentrations were determined using a Bradford Protein Assay (Bio-Rad). 2x Laemmli buffer (Bio-Rad) and 10% β-mercaptoethanol was added to samples. Half of these homogenates were flash frozen in liquid nitrogen and returned to -80°C. The remaining crude homogenate was heated to 70°C for 15 min and 16.0 µg total protein was subjected to Western blotting as described in the Methods. The PDVF membranes were incubated overnight on a shaker (4°C) with anti-ayRhp1 antibodies (0.216 μg/ml), primary antibody with 400x excess peptide on a molar base ('pre-absorption control'), or anti-Tubulin mouse monoclonal antibody (4.7 ng/mL, 12G10 anti-alpha-tubulin, developed by J. Frankel and E.M. Nelsen, and obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242). PVDF membranes were processed as described in the Methods, using goat antirabbit-HRP or goat anti-mouse-HRP diluted 1:5,000 (Bio-Rad) as the secondary antibodies. The anti-ayRhp1 antibodies labeled a ~180 kDa band in ayRhp1-expressing oocytes which was absent in control oocytes and in the pre-absorption control. This band corresponds to ayRhp1 trimers. (Fig. S6A).
The following day, the refrozen ayRhp1-expressing and control oocyte homogenates were thawed to promote dissociation of ayRhp1 trimers, mixed with 2x Laemmli buffer (Bio-Rad) and β-mercaptoethanol, heated to 70°C for 15 min, and subjected to Western blotting and imaging as described above. The anti-ayRhp1 antibodies labeled ~120 and ~60 kDa bands that are absent in the control oocytes and in the pre-absorption control. These bands correspond to ayRhp1 dimers and monomers, respectively. (Fig. S6B-C). In addition to confirming ayRhp1 expression in Xenopus oocytes, these results conform the specificity of the anti-ayRhp1 antibodies against ayRhp1.
Using methods adapted from (84,49), isolated A. yongei cells were prepared by submerging a nubbin in 0.2 μm filtered seawater and brushed with a toothbrush for 1 min. Cells were collected via centrifugation (3,000 x g, 4 minutes, 4°C) and fixed by resuspension in S22 buffer with 4% paraformaldehyde for 15 min on ice. Fixative was removed via centrifugation (3,000 x g, 4 minutes, 4°C) and cells were resuspended in ~500µL of S22 buffer. Cells were then pipetted onto glass microscope slide and allowed to air dry at 4°C for no longer than 1 h before proceeding to immunolabeling.
Tissue sections and isolated cells were blocked for 1 h at room temperature in blocking buffer (4 ml PBS-TX, 80 µl normal goat serum, 0.8 µl keyhole limpet hemocyanin solution), followed by overnight incubation (4°C) with anti-ayRhp1 antibodies (2.16 μg/ml), anti-ayRhp1 antibodies pre-absorbed with excess peptide (8.64 μg/ml) or pre-immune serum (2.16 μg/ml) (all in blocking buffer) (Fig. S4D). Slides were washed in PBS-TX to remove unbound anti-ayRhp1 antibodies (3 x 5 min). Secondary antibodies (goat anti-rabbit-Alexa Fluor555, 4 μg/ml in blocking buffer; Invitrogen) were then added for 1 h at room temperature followed by DAPI DNA Stain (1 μg/ml; Invitrogen) for 5 min at room temperature. Slides were again washed PBS-TX to remove unbound secondary antibody (3 x 5 min) and samples were imaged using a fluorescence microscope (Zeiss AxioObserver, Carl Zeiss AG, Oberkochen, Germany). Figure 1: Maximum likelihood tree of the Acropora yongei Rhesus protein (ayRhp1) in relation to invertebrate and vertebrate Rh proteins. Rh family subgroups are denoted by color: Rh30 (yellow), Rhag (blue), Rhbg (orange), Rhcg (green), Rhp1 (light and dark purple), and Rhp2 (grey) as per (2). Cnidarian Rh proteins are highlighted within the Rhp1 subgroup (light purple). Amino acid sequences were aligned using MUSCLE and a maximum likelihood tree was generated using RAxML (500 bootstraps, PROTGAMMA model of rate heterogeneity, GTR substitution model). Sequences and the outgroup (Nitrosoma europaea Rh) are sourced from (2) with additional sequences identified by NCBI BLAST search. Sequence accession numbers are provided in Supplementary Table 1. Figure 2: Alignment of Acropora yongei Rhesus protein (ayRhp1) with Homo sapiens RhCG (NP_001307970.1). Conserved (blue) and mismatched (orange) hydrophobic channel-lining residues, conserved (green) and mismatched (magenta) cytosolic shunt residues, N-glycan site (black filled triangles), CO2 binding pocket (black outlined triangles) as discussed in (35), Phe-gate (red filled triangles), twin-Histidines (*), and the highly-conserved aspartate residues ($) as discussed in (20,34) are marked. Gaps in sequences are denoted by black dots. The 12 transmembrane helices (TM), as predicted by TMHMM 2.0 (5, 6), for RhCG (20) and ayRhp1 are labeled with spirals.  (B1, D1, E1-I1) Cells displaying ayRhp1 in the symbiosome membrane; notice the separation of host and algal nuclei by ayRhp1. (D1, F1-N1) Cells displaying non-symbiosomal ayRhp1; notice ayRhp1 signal exterior to the host nucleus and absent between the host and algal nuclei. (A2-N2) Corresponding images without brightfield differential interference contrast. Blue arrowheads mark nuclei of host cells; violet arrowheads mark ayRhp1 symbiosome localization. Nuclei are shown in blue and ayRhp1 in green. Scale bars = 5 µm. Figure 6: Western Blot validation of ayRhp1 expression in Xenopus oocytes. (A) Western Blot of once-thawed ayRhp1-expressing (Rh+) and control (C) oocytes, which were homogenized and heated at 70 o C for 15 min before loading into the gels. PVDF membranes were incubated with anti-ayRhp1 antibodies, anti-ayRhp1 antibodies pre-incubated with excess peptide (pre-absorption control), or anti-Tubulin antibody (12G10 anti-alpha-tubulin, developed by J. Frankel and E.M. Nelsen, and obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242).The anti-ayRhp1 antibodies labeled a ~180 kDa band in Rh+ oocytes which was absent in C oocytes (marked with three green asterisks) and in the preabsorption control. This band corresponds to ayRhp1 trimers. The antibodies additionally labeled a ~29 kDa band in both Rh+ and C oocytes (marked with a black asterisk) which was not present in the pre-absoprtion control; this band represents off-target recognition of Xenopus protein(s). The anti-tubulin antibody labeled a ~50 kDa protein in both treatments demonstrating equivalent protein concentration in Rh+ and C samples. (B, C) Western Blots of rethawed Rh+ (B) and C (C) oocyte homogenates which were heated at 70 o C for 15 min before loading into the gels. The anti-ayRhp1 antibodies labeled a ~120 kDa band (marked with two red asterisks) and a ~60 kDa band (marked with one red asterisk) that are absent in the C oocytes and in the pre-absorption control. These bands correspond to ayRhp1 dimers and monomers, respectively. The ~29 kDa band is still present in the rethawed homogenates. Table 1: NCBI accession numbers for sequences used in phylogenetic analysis (Fig. S1).

SI Files
Data S1 Cell count data used for determining diel ayRhp1 localization in coral host cells.