Structure and function of LCI1: a plasma membrane CO2 channel in the Chlamydomonas CO2 concentrating mechanism

Abstract

Microalgae and cyanobacteria contribute roughly half of the global photosynthetic carbon assimilation. Faced with limited access to CO₂ in aquatic environments, which can vary daily or hourly, these microorganisms have evolved use of an efficient CO₂ concentrating mechanism (CCM) to accumulate high internal concentrations of inorganic carbon (C_i ) to maintain photosynthetic performance. For eukaryotic algae, a combination of molecular, genetic and physiological studies using the model organism Chlamydomonas reinhardtii, have revealed the function and molecular characteristics of many CCM components, including active C_i uptake systems. Fundamental to eukaryotic C_i uptake systems are C_i transporters/channels located in membranes of various cell compartments, which together facilitate the movement of C_i from the environment into the chloroplast, where primary CO₂ assimilation occurs. Two putative plasma membrane C_i transporters, HLA3 and LCI1, are reportedly involved in active C_i uptake. Based on previous studies, HLA3 clearly plays a meaningful role in HCO₃^- transport, but the function of LCI1 has not yet been thoroughly investigated so remains somewhat obscure. Here we report a crystal structure of the full-length LCI1 membrane protein to reveal LCI1 structural characteristics, as well as in vivo physiological studies in an LCI1 loss-of-function mutant to reveal the C_i species preference for LCI1. Together, these new studies demonstrate LCI1 plays an important role in active CO₂ uptake and that LCI1 likely functions as a plasma membrane CO₂ channel, possibly a gated channel.

Keywords: Chlamydomonas; CCM; CO2; Ci; LCI1; channel; photosynthesis; transporter.

Figure 1.

Structure of the Chlamydomonas reinhardtii LCI1. (A) Ribbon diagram of an LCI1 monomer viewed in the membrane plane. The molecule is colored using a rainbow gradient from the N-terminus (blue) to the C-terminus (red). (B) Ribbon diagram of an LCI1 trimer viewed in the membrane plane. The three protomers are colored green, red and yellow, respectively. The transmembrane segments (TMs) and α-helices (αs) of the front protomer (green) of LCI1 are labeled. The Figure was prepared using PyMOL (http://www.pymol.sourceforge.net).

Figure 2.

(A) Each LCI1 subunit forms a channel (colored green) that spans approximately two-thirds of the transmembrane. The radius of the channel was calculated using the program HOLE (Smart et al., 1993). The channel is closed due to interaction between the sidechains of residues E87 and N161. (B) The LCI1 channel (colored pink) is surrounded by 22 amino acids, including V26, A30, L33, L42, Y52, Y56, F60, I66, F77, Q80, I83, I84, N125, A128, T137, V141, F142, I147, A150, L154 and V157, and containing a potential CO₂-binding site located right below residues E87 and N161. This channel representation was generated using the program CAVER (http://loschmidt.chemi.muni.cz/caver). The secondary structural elements of the LCI1 protomer are in gray, and residues involved in forming this channel, along with residues E87 and N161, are represented by green sticks.

Figure 3.

Electrostatic surface potentials of LCI1. Surface representations of the inside (A), top view (B) and bottom view (C) of the LCI1 channel colored by charge (red; negative −15 kT/e, blue; positive +15 kT/e).

Figure 4.

Putative CO₂ binding site. (A) Stereo view of the F_o - F_c electron density map of bound ligand, presumed to be CO₂, in LCI1, with the putative bound CO₂ ligand and residues putatively involved in CO₂ binding shown as a stick model (cyan, carbon; blue, nitrogen) and as green sticks, respectively. The F_o - F_c map is contoured at 3.0 σ (blue mesh). (B) A composite figure showing the locations of the predicted bound CO₂ ligand (yellow) and the putative bound CO₂ (cyan) identified in the LCI1 crystal structure.

Figure 5.

Mass spectrum of LCI1 obtained under native conditions. The spectra indicate the presence of the LCI1 trimer (purple circles) and its charge state series. The measured mass of this trimer is 66113.01 ± 0.27 Da, which is in good agreement with the theoretical mass of the trimeric LCI1 protein (66115.8 Da) without the first methionine residue on each monomer. The other species (cyan, yellow and red triangles) correspond to the lipid bound trimers, and these are endogenously purified. Lipid analysis on this sample revealed the presence of PA(16:0/18:1) and PA(18:1/18:1) as shown in Figure S2, which are bound in different numbers and combinations. All the molecular masses observed are listed in Table S2.

Figure 6.

Steered molecular dynamics (SMD) simulations of the migration of CO₂ and HCO₃⁻ through LCI1. (A) CO₂ trajectory through LCI1 in SMD is shown as blue mesh. LCI1 is oriented with the channel along the z-axis and the position of ligand along the channel is measured as distance from the protein center of mass along the z-axis. (B) Plots of applied forces as a function of ligand positions along the channel (blue, CO₂; red, HCO₃⁻), which indicate that pushing HCO₃⁻ through the channel requires much larger force, suggesting that CO₂ may be the preferred ligand. A black rectangle highlights the region corresponding to the putative CO₂-binding site. The two peaks marked in the CO₂ plot (blue) indicate local electrostatic interactions between (C) putative bound CO₂ and residues E87 and N161; and (D) putative bound CO₂ and residue Q16. (E) Root mean square fluctuations (RMSF) of the LCI1 residues during the SMD simulations with CO₂ (blue) and HCO₃⁻ (red).

Figure 7.

Uncompensated contribution of LCI1 (C, D), LCIB (E, F), and LCIA (G, H) in a wild-type background at pH 6 (open squares), pH 7.3 (closed squares) and pH 9 (red circles) plotted against calculated CO₂ concentration (left) and HCO₃⁻ concentration (right). The CO₂ and HCO₃⁻ concentration ranges covered are 0–25 μM and 0–120 μM, respectively. A and B are the original O₂ evolution rates of the wild type. Note that the Y-axis scales on the original O₂ evolution plots (A, B) are different than those on the uncompensated contribution plots (C–H). Uncompensated contributions were calculated by subtracting O₂ evolution rates of the single mutant from wild type strain (LCI1 in wild type = 21gr-lci1; LCIB in wild type = 21gr-pmp1; LCIA in wild type = 21gr-lcia).

Figure 8.

Uncompensated contribution of LCI1 (C, D), LCIB (E, F), and LCIA (G, H) in a wild-type background at pH 6 (open squares), pH 7.3 (closed squares) and pH 9 (red circles) plotted against the calculated CO₂ concentration (left) and HCO₃⁻ concentration (right). The CO₂ and HCO₃⁻ concentration ranges covered are 0–250 μM and 0–1200 μM, respectively. A and B are the original wild-type O₂ evolution rates. Note that the Y-axis scales on the original O₂ evolution plots (A, B) are different than those on the uncompensated contribution plots (C–H). Uncompensated contributions in wild type were calculated by subtracting O₂ evolution rates of the single mutant from wild type strain (LCI1 in wild type = 21gr-lci1; LCIB in wild type = 21gr-pmp1; LCIA in wild type = 21gr-lcia).

Figure 9.

Uncompensated contributions of LCI1 (B, E) and LCIB (C, F) in an lcia mutant background at pH 6 (open squares), pH 7.3 (closed squares) and pH 9 (red circles) plotted against calculated CO₂ concentration (A–C) and HCO₃⁻ concentration (D–F). The CO₂ and HCO₃⁻ concentration ranges covered are 0–25 μM and 0–120 μM, respectively. A and D are the original lcia mutant O₂ evolution rates. Note that the Y-axis scales on the original O₂ evolution plots (A, D) are different than those on the uncompensated contribution plots (B, C, E, F). Uncompensated contributions were calculated by subtracting O₂ evolution rates of the mutant from its background strain: LCI1 in lcia = lcia-l1a; LCIB in lcia = lcia-lab. l1a is the LCI1/LCIA double mutant and lab is the LCIA/LCIB double mutant

Figure 10.

Uncompensated contributions of LCI1 (A, C) and LCIA (E, F) in a pmp1 strain (lcib mutant background) at pH 6 (open squares), pH 7.3 (closed squares) and pH 9 (red circles) plotted against the calculated CO₂ concentration (A, E) and the HCO₃⁻ concentration (C, F). The CO₂ and HCO₃⁻ concentration ranges covered are 0–25 μM and 0–120 μM, respectively. B and D are the original pmp1 mutant O₂ evolution rates. Note that the Y-axis scales on the original O₂ evolution plots (B, D) are different than those on the uncompensated contribution plots (A, C, E, F). Uncompensated contributions were calculated by subtracting the O₂ evolution rates of the mutant from its background strain: LCI1 in pmp1 = pmp1-l1b; LCIA in pmp1 = pmp1-lab. l1b is the LCI1/LCIB double mutant and lab is the LCIA/LCIB double mutant.

Figure 11.

Percentage of LCIA (A, C) and LCI1 (B, D) uncompensated contribution to photosynthesis in a pmp1 mutant background at pH 6 (open triangles), pH 7.3 (closed triangles) and pH 9 (red diamonds) plotted as a function of calculated CO₂ concentrations of 0–25 μM (A, B) and of calculated HCO₃⁻ concentrations of 0–120 μM (C, D).

Figure 12.

Calculated, uncompensated contribution of LCI1 (A, B), LCIA (C, D), and LCIB (E, F) at pH 6 (left) and at pH 7.3 (right) in a wild-type background (black triangles), a pmp1 mutant background (red triangles), an lcia background (green triangles), and an lci1 mutant background (blue triangles). CO₂ concentration was calculated from total C_i assuming CO₂ and HCO₃⁻ are in equilibrium. Uncompensated contributions were calculated by subtracting the O₂ evolution rates of the mutant from those of its background strain (LCI1 in wild type = 21gr-lci1; LCI1 in pmp1 = pmp1-l1b; LCI1 in lcia = lcia-l1a; LCIB in wild type = 21gr-pmp1; LCIB in lcia = lcia-lab; LCIB in lci1 = lci1-l1b; LCIA in wild type = 21gr-lcia; LCIA in lci1 = lci1-l1a; LCIA in pmp1 = pmp1-lab). l1b is the LCI1/LCIB double mutant; lab is the LCIA/LCIB double mutant; l1a is the LCI1/LCIA double mutant.