DOI: 10.5061/dryad.m0cfxpp7m
title: Data from: Branchial CO2 and ammonia excretion in crustaceans: Involvement of an apical Rhesus-like glycoprotein

description: Aim To determine whether the crustacean Rh1 protein functions as a dual
 CO2/ammonia transporter and investigate its role in branchial ammonia
 excretion and acid-base regulation. Methods Sequence analysis of decapod
 Rh1 proteins was used to determine the conservation of amino acid residues
 putatively involved in ammonia transport and CO2 binding in human and
 bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a
 representative of decapod Rh1 proteins, we test the ammonia and CO2
 transport capabilities of CmRh1 through heterologous expression in yeast
 and Xenopus oocytes coupled with site-directed mutagenesis. Quantitative
 PCR was used to assess distribution of CmRh1 mRNA in various tissues.
 Western blotting was used to assess CmRh1 protein expression changes in
 response to high environmental ammonia and CO2. Further,
 immunohistochemistry was used to assess sub-cellular localization of CmRh1
 and a membrane bound carbonic anhydrase (CmCAg). Results Sequence analysis
 of decapod Rh proteins revealed high conservation of several amino acid
 residues putatively involved in conducting ammonia transport and CO2
 binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and
 CO2 transport which was nullified in CmRh1 D180N mutant oocytes. Transport
 of the ammonia analogue methylamine by CmRh1 is dependent on both ionized
 and un-ionized ammonia/methylamine species. CmRh1 was co-localized with
 CmCAg to the apical membrane of the crustacean gill and only experienced
 decreased protein expression in the anterior gills when exposed to high
 environmental ammonia. Conclusion CmRh1 is the first identified apical
 transporter-mediated route for ammonia and CO2 excretion in the crustacean
 gill. Our findings shed further light on the potential universality of
 dual ammonia and CO2 transport capacity of Rhesus glycoproteins in both
 vertebrates and invertebrates.
description: # Branchial CO2 and ammonia excretion in crustaceans: Involvement of an
 apical Rhesus-like glycoprotein This data set provides data collected from
 4 experiments. Experiment 1:Gene expression of CmRh1 across different
 tissues and Protein expression in gills during exposure to high
 environmental ammonia and hypercapnia Experiment 2:Time dependent
 radiolabelled methyl amine uptake in Xenopus oocytes expressing CmRh1,
 CmRh1 D180N variant and water-injected sham Experiment 3: Effect of
 environmental ammonia, methyl amine concentration, and pH on radiolabelled
 methyl amine uptake in Xenopus oocytes expressing CmRh1, CmRh1 D180N
 variant and water-injected sham Experiment 4: CO2 excretion rate in
 Xenopus oocytes expressing CmRh1, CmRh1 D180N variant and water injected
 sham ## Description of the data and file structure Figure 2A data is
 organized by gene assayed, sample, tissue type, treatment, starting
 quantity, and then a normalized gene expression comparing CmRh1 (gene of
 interest) to normalizing house keeping gene (arginine kinase or ribosomal
 protein S3). N/A indicates that there was no detectable expression for
 that particular sample. Figure 2C+D data is organized by sample, tissue
 type, treatment, and relative protein expression of CmRh1. N/A indicates a
 missing protein sample for the particular individual. Figure 4A data is
 organized by what was injected into the oocyte (water=sham, CmRh1 cRNA, or
 CmRh1 D180N variant cRNA), the incubation time, and the dependent variable
 which was rate of methyl amine uptake. N/A indicates an oocyte had burst
 prior to scintillation counting result in no reliable data for that
 replicate. Figure 4B+C data is organized by what was injected into the
 oocyte (water=sham, CmRh1 cRNA, or CmRh1 D180N variant cRNA), the ovary
 used (1 or 2), the concentration of unlabelled methyl amine,
 pH,concentration of ammonium chloride, and the dependent variable which
 was rate of methyl amine uptake, and the final column reflects methyl
 amine uptake rate with subtraction of the average sham rate for each
 group. N/A indicates an oocyte had burst prior to scintillation counting
 result in no reliable data for that replicate or sham subtraction rate is
 not applicable for the particular sample. Figure 5 data is organized by
 what was injected into the oocyte (water=sham, CmRh1 cRNA, or CmRh1 D180N
 variant cRNA) and the CO2 excretion rate measured. N/A indicates that the
 oocyte had burst resulting in no reliable measurement for that replicate.