How protein - protein interactions contribute to pyrenoid formation in Chlamydomonas.

The chloroplast pyrenoid, an important component of the CO

The physiological consequences of this packaging of Rubisco are profound. In general, algae with pyrenoids have a much higher affinity for inorganic carbon (Ci = CO2 + HCO3- + CO3-2) than terrestrial C3 plants. These algae are able to raise the CO2 concentration for Rubisco through the CO2 concentrating mechanism (CCM). Our current thinking is that the packaging of Rubisco is a requirement for the CCM (Mackinder, 2018; Moroney and Ynalvez, 2007; Spalding, 2008). However, since CO2 can readily cross cell membranes (Tolleter ), how can a single-celled organism possibly concentrate CO2? Current CCM models have cells accumulating HCO3-, an ion that does not readily cross membranes, instead of CO2 directly. After taking up HCO3-, a key step in this process is the conversion of the accumulated HCO3- to CO2 by the action of the enzyme carbonic anhydrase (CA). This raises the CO2 concentration at Rubisco, which is located physically close to the CA. Then the Rubisco has a chance to fix the substrate CO2 before the CO2 diffuses away (Box 1A). If the pyrenoid is not correctly formed, the CO2 will inevitably leak out of the cell (Box 1B). Another proposed reason for the pyrenoid organization is that it facilitates the recapture of some of the CO2 as it leaks past Rubisco. In Chlamydomonas, the pyrenoid is surrounded by a starch sheath and the heteromeric protein LCIB/LCIC. The LCIB/C complex has been proposed to act as a CA, converting leaking CO2 to the less permeant HCO3-. Thus, CO2 generated by the CCM must pass through Rubisco and then a CA layer before it has a chance to leave the chloroplast (Box 1A).

How the pyrenoid reduces CO2 leakage in Chlamydomonas reinhardtii.A) In a functional pyrenoid, a starch sheath forms around the pyrenoid, which contains most of the Rubisco (indicated with purple). Rubisco interacts with EPYC1 (shown in blue) and that interaction aids in the formation of the pyrenoid. In this figure HCO3- uptake and its subsequent conversion to CO2 with the help of carbonic anhydrase has not been shown for the sake of simplicity. The Rubisco product glycerate-3-phosphate (PGA), shown by purple arrows, forms when CO2 concentrates in the pyrenoid. CO2 that leaks past Rubisco is sometimes recaptured and converted to HCO3- (indicated by green arrows) by LCIB/C (red), a stromal carbonic anhydrase, or exits the chloroplast entirely (dotted arrows). B) Absence of EPYC1 prevents the formation of a pyrenoid and accumulated HCO3- once converted to CO2 easily leaks out and is not recaptured.

In Chlamydomonas, the protein sequence of the Rubisco small subunit (SSU) and the protein EPYC1 (essential pyrenoid component 1) are important to the formation of the pyrenoid (Mackinder ; Meyer ). The first evidence came from the work of Meyer , who showed that Chlamydomonas cells expressing the Arabidopsis SSU instead of the Chlamydomonas SSU failed to form a pyrenoid and failed to grow in ambient CO2. However, it was interesting that the Rubisco in these cells, consisting of the Chlamydomonas large subunit (LSU) and Arabidopsis SSU, was still enzymatically active. Meyer also identified regions in the Rubisco SSU necessary for pyrenoid formation. Replacing specific Chlamydomonas SSU α-helices with the corresponding sequence from plant Rubisco SSU resulted in cells without pyrenoids and with defective CCMs. In 2016, Mackinder et al. found that EPYC1 was also required for pyrenoid formation. EPYC1 is a linker protein which binds to Rubisco in Chlamydomonas and facilitates the liquid-like formation (Küken ; Rosenzweig ). It is not present in terrestrial plants. Loss of EPYC1 results in Chlamydomonas cells being unable to form a normal pyrenoid and develop a functional CCM (Mackinder ).

The work described by Atkinson greatly extends these studies by investigating which regions of each protein are required for SSU-EPYC1 binding. Chlamydomonas has two genes encoding the SSUs, designated S1Cr and S2Cr. Atkinson used quantitative yeast two-hybrid (Y2H) experiments to show that EPYC1 strongly interacts with both Chlamydomonas SSU homologs but not with the Arabidopsis SSU (1AAt). They then systematically replaced parts of the Chlamydomonas SSU with the corresponding regions of the Arabidopsis SSU. They found that multiple parts of the Chlamydomonas SSU contributed to the SSU-EPYC1 interactions. Substituting in the two α-helices from the Chlamydomonas SSU into the Arabidopsis SSU was essential for interaction, while adding in the β sheets or the βA-βB loop region greatly increased SSU-EPYC1 binding. Atkinson repeated these Y2H experiments but with modifications of EPYC1. EPYC1 has four repeat regions with short terminus regions. They found that each of the repeated regions and the C terminus contributed to the binding of EPYC1 to the SSU. Thus, large portions of each protein were important to the strength of the protein-protein interaction. They also found that a mixture of EPYC1 and the Chlamydomonas SSU could phase separate and form liquid droplets in vitro, indicating that a large number of components may not be needed to form a pyrenoid-like structure.

The question arises: can researchers reconstruct a pyrenoid in higher plants? Photosynthesis modelling suggests that introducing algal CCM bicarbonate transporters into C3 plants and packaging Rubisco could lead to a significant increase in photosynthetic efficiency (Furbank ; McGrath and Long, 2014; Zhu ). Atkinson took a significant step towards building a pyrenoid by expressing EPYC1 in Arabidopsis wild type plants as well as Arabidopsis plants expressing the Chlamydomonas SSU. However, no Rubisco aggregation was seen in either; instead, an even distribution of Rubisco was seen throughout the chloroplast. It was encouraging that they were able to express EPYC1 in plants, although they postulate that the amount of EPYC1 present was still too low to expect liquid phase separation to occur, which is due to EPYC1-Rubisco interactions. An in vitro assay developed by Wunder showed that a critical EPYC1:Rubisco ratio is required before phase separation occurs; the EPYC1 producing plants in this paper may not have enough of the linker protein.

These results indicate that with a higher EPYC1: Rubisco expression, a packaging of Rubisco might be engineered in a C3 plant, as EPYC1 doesn’t appear to need additional proteins to bind to Rubisco in vitro. However, in vivo, other components might be required (Ma ). Mackinder showed that in Chlamydomonas, EPYC1 interacts with a protein kinase and 14-3-3 proteins, suggesting a role of phosphorylation in Rubisco-EPYC1 assembly. This finding fits with earlier studies showing that EPYC1 is a phosphoprotein (Turkina ). The Atkinson paper highlights the residues needed for EPYC1-Rubisco interaction in order to obtain a liquid-like pyrenoid. Thus, both EPYC1 and modified Rubiscos have been successfully expressed in heterologous systems of yeast and Arabidopsis by Atkinson to show that the strength of the EPYC1-Rubisco interaction can be manipulated. This is a big step towards the end goal of organizing Rubisco in C3 plants into a pyrenoid-like structure. Rubisco organized in this fashion should be more efficient at fixing CO2 if a functional CCM is introduced into C3 plants. Rubisco in a pyrenoid-like structure should better capture CO2 generated by a CCM thus preventing CO2 leakage (Box 1A).