Co-option of a conserved host glutamine transporter facilitates aphid/ Buchnera metabolic integration

Abstract

Organisms across the tree of life colonize novel environments by partnering with bacterial symbionts. These symbioses are characterized by intimate integration of host/endosymbiont biology at multiple levels, including metabolically. Metabolic integration is particularly important for sap-feeding insects and their symbionts, which supplement nutritionally unbalanced host diets. Many studies reveal parallel evolution of host/endosymbiont metabolic complementarity in amino acid biosynthesis, raising questions about how amino acid metabolism is regulated, how regulatory mechanisms evolve, and the extent to which similar mechanisms evolve in different systems. In the aphid/Buchnera symbiosis, the transporter ApGLNT1 (Acyrthosiphon pisum glutamine transporter 1) supplies glutamine, an amino donor in transamination reactions, to bacteriocytes (where Buchnera reside) and is competitively inhibited by Buchnera-supplied arginine-consistent with a role regulating amino acid metabolism given host demand for Buchnera-produced amino acids. We examined how ApGLNT1 evolved a regulatory role by functionally characterizing orthologs in insects with and without endosymbionts. ApGLNT1 orthologs are functionally similar, and orthology searches coupled with homology modeling revealed that GLNT1 is ancient and structurally conserved across insects. Our results indicate that the ApGLNT1 symbiotic regulatory role is derived from its ancestral role and, in aphids, is likely facilitated by loss of arginine biosynthesis through the urea cycle. Given consistent loss of host arginine biosynthesis and retention of endosymbiont arginine supply, we hypothesize that GLNT1 is a general mechanism regulating amino acid metabolism in sap-feeding insects. This work fills a gap, highlighting the broad importance of co-option of ancestral proteins to novel contexts in the evolution of host/symbiont systems.

Keywords: A. pisum; Buchnera; amino acid metabolism; gene co-option; symbiosis.

Fig. 1.

Aphids house Buchnera in bacteriocyte cells and ApGLNT1 localizes to the bacteriocyte membrane. (A) Confocal microscope image of a bacteriocyte from a three-day-old asexual female aphid. DNA is stained in DAPI (white color). Bacteriocytes make up the symbiotic organ located in the aphid’s abdomen. (B) Drawing of an aphid showing the bacteriome, the symbiotic organ made up of bacteriocytes (outlined in green). (C) Confocal microscope image of ApGLNT1 immunolocalization to the plasma membrane of a bacteriocyte from an asexual female adult aphid. ApGLNT1 is shown in green and DNA is stained with DAPI (blue). The nucleus in each bacteriocyte image is marked with an “n”. (Scale bars are 10 µm.)

Fig. 2.

Phylogenetic framework and taxon sampling. Phylogenetic relationships among insects sampled in this study for functional characterization and/or urea cycle gene annotation. In addition to A. pisum, we functionally characterized ApGLNT1 orthologs from two sap-feeding insects (Planococcus citri and Diaphorina citri), one blood-feeding insect (Pediculus humanus), and one insect without an intracellular symbiont (Tribolium castaneum). Bemisia tabaci was included only in our annotation of urea cycle genes. Insect names appear at the end of each branch, with the names of intracellular bacterial symbionts (if any) listed below. The sap-feeding taxa (P. citri, B. tabaci, and D. citri) represent superfamilies of the hemipteran suborder Sternorrhyncha (highlighted by the box), to which aphids belong. The evolution of sap-feeding is mapped onto the tree with an arrow. Relationships shown here are based on published phylogenetic studies (–30).

Fig. 3.

ApGLNT1 orthologs induce an inward current in response to glutamine that is dependent upon extracellular pH. Graphs show mean ± SE of response amplitudes to 5 mM glutamine at pH 8.5, 7.5, 6.5, and 5.5 normalized to the mean response amplitude at pH 5.5. Letters above bars in each graph mark statistically significant differences based on a Friedman test [ApGLNT1: χ²(2)= 17, P < 0.0001; PcitGLNT1: χ²(2) = 11.10, P = 0.0009; DcitGLNT1: χ²(2) = 18.77, P = 0.0003; PhumGLNT1: χ²(2) = 21.75, P < 0.0001; TcasGLNT1: χ²(2) = 33.30, P < 0.0001]. Representative current recordings for pH dependence runs are shown in SI Appendix, Fig. S1, along with representative current recordings for control, water-injected oocytes. Sample sizes are given in SI Appendix, Table S2.

Fig. 4.

GLNT1 orthologs are low-affinity glutamine transporters. Glutamine-induced inward current at different concentrations of glutamine and EC₅₀ values for GLNT1 orthologs from (A) A. pisum, P. citri, D. citri, and T. castaneum and (B) P. humanus. P. humanus was plotted separately from the other species because the scale on the horizontal axis is different. Response amplitudes were normalized and concentration–response analysis performed as described in Methods. Points on each graph represent means ± SE. Sample sizes are given in SI Appendix, Table S2. Representative current recordings for glutamine response and control runs with water-injected oocytes are shown in SI Appendix, Fig. S2.

Fig. 5.

GLNT1 orthologs have narrow substrate selectivity. Representative current recordings for amino acid screens in GLNT orthologs and water-injected oocytes (controls). Black bars above the water-injected oocyte traces for ApGLNT1 and DcitGLNT1 runs mark the timing and duration of amino acid applications. Results shown in representative current responses were qualitatively obtained in 100% of experimental runs with slight quantitative differences in response amplitudes. Amino acids were applied at 2 mM to oocytes expressing ApGLNT1, PcitGLNT1, PhumGLNT1, and TcasGLNT1 and at 10 mM to oocytes expressing DcitGLNT1 (because of low sensitivity), with the exception of tyrosine, which was applied at 2.5 mM because it is insoluble at higher concentrations. Sample sizes are given in SI Appendix, Table S2.

Fig. 6.

Glutamine transport is inhibited by arginine in GLNT1 orthologs. Arginine inhibition curves and estimated IC₅₀ values for each GLNT1 ortholog. Arginine inhibition of glutamine response was measured by applying a mixture of glutamine at EC₅₀ plus increasing concentrations of arginine. Responses to glutamine + arginine were normalized to the preceding response to glutamine alone. Concentration inhibition analysis was performed as described in Methods. Points on each graph represent mean ± SE. N values for each transporter are given in SI Appendix, Table S2. Representative current recordings for arginine inhibition and control, water-injected oocytes are shown in SI Appendix, Fig. S3.

Fig. 7.

Conservation of substrate binding pocket residues in GLNT1 orthologs. (A and B) Homology model of ApGLNT1 created using HHPred and Modeller superimposed on to the crystal structure of bacterial amino acid transporter GkApcT (PDB 5OQT, gray) from which it was derived. ApGLNT1 is shown as cyan with TM domains 3 and 8 shown in blue. Key residues C198 (TM3, blue) and Q383 (TM8, blue) are shown as sticks. Glutamine (gray sticks and spheres) is shown as a substrate in the binding pocket (positioned using crystal-bound alanine in GkApcT). C198 and Q383 line the deep areas of the substrate binding pocket and are adjacent to the substrate side chain. In (A), proteins are shown “side on”, as they would reside in the membrane and in (B), “top down”, above the plane of the membrane, looking down into the binding pocket. (C and D) Models of GLNT1 orthologs produced by AlphaFold (models are calculated without substrate). The overlaid models are from functionally characterized and more distant taxa as follows: ApGLNT1 (cyan), DcitGLNT1 (magenta), PcitGLNT1 (purple), PhumGLNT1 (orange), TcasGLNT1 (green); (D) ApGLNT1 (cyan), CsplGLNT1 (lilac) and Dmel (D. melanogaster) CG43693 (yellow). The models show conservation of a Cys residue, in the helix of TM3, in the equivalent position to C198 in ApGLNT1 and a Gln residue, in the helix of TM8, in the equivalent position to Q383 in ApGLNT1. In all figures, the intracellular N-terminal section has been omitted for clarity. Four-letter taxon IDs for sequences are listed in Dataset S1.

Fig. 8.

Key positions within TM3 and TM8 are conserved in GLNT1 orthologs. Alignments of central portions of TM3 and TM8 from GLNT1-related sequences taken from whole protein alignments of various insect taxa. The degree of homology is denoted by shading. Residues in the equivalent position to ApGLNT1 C198 and Q383 are shown in highlighted colors. Phylogeny to the left of the alignment shows relationships between orthologs in the GLNT1 clade. It is part of the full maximum likelihood phylogeny of the insect SLC36 transporter family depicted in SI Appendix, Fig. S6. Four-letter taxon IDs for sequences are listed in Dataset S1. Numbers following taxon IDs for auchenorrhynchans (Nlug, Lstr, Pspu, and Dsem) indicate designated paralog number.