Rubisco Small-Subunit Α-Helices Control Pyrenoid Formation in Chlamydomonas
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Rubisco small-subunit α-helices control pyrenoid formation in Chlamydomonas Moritz T. Meyera,1,2, Todor Genkovb,2,3, Jeremy N. Skepperc, Juliette Jouheta,4, Madeline C. Mitchella, Robert J. Spreitzerb, and Howard Griffithsa aDepartment of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom; bDepartment of Biochemistry, University of Nebraska, Lincoln, NE 68588; and cDepartment of Physiology, University of Cambridge, Cambridge CB2 3DY, United Kingdom Edited by George H. Lorimer, University of Maryland, College Park, MD, and approved September 21, 2012 (received for review June 27, 2012) The pyrenoid is a subcellular microcompartment in which algae and mechanistic properties of such biochemical CCM processes sequester the primary carboxylase, ribulose-1,5-bisphosphate car- could be a prelude to their introduction into staple crops such as boxylase/oxygenase (Rubisco). The pyrenoid is associated with rice (6). aCO2-concentrating mechanism (CCM), which improves the oper- In parallel, we also need to learn from cyanobacteria and ating efficiency of carbon assimilation and overcomes diffusive eukaryotic algae, which currently sequester an equivalent pro- Chla- limitations in aquatic photosynthesis. Using the model alga portion of anthropogenic CO2 emissions. In aquatic photosyn- mydomonas reinhardtii , we show that pyrenoid formation, Rubisco thetic microorganisms, CO2 assimilation is supported by a range aggregation, and CCM activity relate to discrete regions of the of biophysical CCMs, which use transmembrane transporters and Rubisco small subunit (SSU). Specifically, pyrenoid occurrence was carbonic anhydrases to concentrate DIC intracellularly by up to shown to be conditioned by the amino acid composition of two 1,000-fold (7), with Rubisco packaged into an associated sub- surface-exposed α-helices of the SSU: higher plant-like helices knock cellular microcompartment. The confinement of Rubisco helps out the pyrenoid, whereas native algal helices establish a pyrenoid. to limit CO2 back-diffusion (CCM “leakage”) inherently associ- We have also established that pyrenoid integrity was essential for ated with the slow turnover rate of the enzyme. Understanding the operation of an active CCM. With the algal CCM being function- the molecular determinants of these biophysical CCM processes, ally analogous to the terrestrial C4 pathway in higher plants, such which improve the operating efficiency of Rubisco, could also pro- insights may offer a route toward transforming algal and higher vide a route toward augmenting crop productivity for the future (8). plant productivity for the future. The cyanobacterial CCM sequesters Rubisco into carboxysomes, which are semipermeable microcompartments surrounded by a algal photosynthesis | carbon fixation | chloroplast | protein engineering protein shell that resembles viral capsids (9). Nearly all eukary- otic algal lineages possess a functionally analogous structure, the he catalytic activities of ribulose-1,5-bisphosphate carboxyl- pyrenoid (10), which is also found in one terrestrial plant group, Tase/oxygenase (Rubisco) are often considered to be flawed, the ancient hornworts (11). In transmission electron micro- inefficient, or at best confused (1, 2), and yet the resultant graphs, pyrenoids appear as unstructured electron-dense com- photosynthetic CO2 assimilation into three-carbon sugars (“C3” partments in the chloroplast stroma, usually traversed by several pathway) sustains life on earth. Served by a single active site, thylakoid membranes. Although pyrenoids seemingly lack a pro- which evolved under anaerobic conditions, Rubisco carboxylase tein coat or membrane, a starch sheath is often deposited at the function became competitively inhibited by O2, a by-product of immediate periphery. The presence of a pyrenoid is associated photosynthetic light reactions. Relatively high atmospheric CO2 with the occurrence of a CCM (12), although the importance of concentrations, in equilibrium with the aquatic dissolved inorganic the pyrenoid in the functional operation of a CCM has hitherto carbon (DIC) pool, continued to sustain Rubisco carboxylase ac- not been demonstrated. The bundle sheath in C4 photosynthesis tivity through the gradually increasing O2 concentration, and be- has a similar role in sequestering Rubisco and containing CO2 yond the great oxidation event at some 2.4 billion years ago (bya). leakage (13). Based on C4 isotopic discrimination models (14) However, between 0.6 and 1 bya, cyanobacterial photosynthesis and species abundance in cyanobacterial and eukaryotic phyto- was limited by inorganic carbon supply to the extent that some plankton assemblages (15, 16), nearly one-half of the 105 peta- form of carbon-concentrating mechanism (CCM) may then have grams of carbon fixed each year (17) is contingent on some form evolved (3). Subsequently, most aquatic eukaryotic lineages also of CCM and Rubisco compartmentation. Whereas the C4 and developed a CCM to suppress oxygenase activity and the associ- CAM biochemical systems, and the cyanobacterial carboxysome, ated wasteful photorespiration, and overcome CO2 limitation due are increasingly well resolved structurally and functionally (9, to slow diffusivity in water (4). When plants conquered land, the role of stomata, and in- ternalization of gas exchange surfaces to minimize liquid phase Author contributions: M.T.M. and T.G. designed research; M.T.M., T.G., J.N.S., J.J., and resistance, allowed CO diffusion to supply carboxylase activity M.C.M. performed research; R.J.S. and H.G. contributed new reagents/analytic tools; and 2 M.T.M. and H.G. wrote the paper. of Rubisco. Despite subsequent variations in atmospheric CO2 The authors declare no conflict of interest. concentrations, for 400 million years C3 terrestrial plants have dominated carbon storage in biomass and soils. Today, they se- This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. quester nearly one-third of anthropogenic CO2 emissions, as well as providing food and fuel for the burgeoning global population, 1To whom correspondence should be addressed. E-mail: [email protected]. 2M.T.M. and T.G. contributed equally to this work. via many staple crops. However, it is thought that CO2 starvation and a changing climate over the past 20–30 million years have 3Present address: Center for Biocatalysis and Bioprocessing, The University of Iowa, Coral- been major selection pressures to develop other mechanisms of ville, IA 52241. 4Present address: Laboratoire de Physiologie Cellulaire Végétale, Unité Mixte de Re- CO2 concentration by terrestrial plants, which tend to use a four- cherche 5168, Centre National de la Recherche Scientifique, Commissariat à l’Energie carbon organic acid currency to power a biochemical CCM in the Atomique, Institut National de la Recherche Agronomique, Université Joseph Fourier, C4 pathway and crassulacean acid metabolism (CAM). Today, in 38054 Grenoble, France. a warming world, as crop yields plateau (5) and the human popu- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lation continues to grow, understanding the fundamental genetic 1073/pnas.1210993109/-/DCSupplemental. 19474–19479 | PNAS | November 20, 2012 | vol. 109 | no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1210993109 Downloaded by guest on September 25, 2021 A (higher plant) SSU display reduced levels of photosynthetic growth and systematically lack a pyrenoid (21), implying a re- lationship between SSU, pyrenoid, and a functional CCM. The Py work in this paper focuses on specific solvent-exposed Rubisco SSU regions, i.e., two external α-helices, based on the hypothesis that Rubisco aggregation within the pyrenoid could be mediated by extrinsic protein interactions. Using a site-directed mutagen- esis approach, we show that these discrete SSU regions condition pyrenoid formation, and that confinement of Rubisco into the pyrenoid is coupled to the operation of an active CCM in Chla- B mydomonas. Such insights may well potentiate the introduction of algal CCM components into higher plants, for which the forma- tion of a microcompartment containing Rubisco, within the chloroplast, may well be an essential prerequisite (8). Results Land Plant Rubisco SSU with Substituted Algal α-Helices Sustains Growth in Vivo in Chlamydomonas Despite Compromised Rubisco (L2)4(S4)2 (L2)4(S4)2 (S4)2 Top Side Side Kinetics in Vitro. Form I Rubisco, which is common to cyano- bacteria, green algae, and land plants, is composed of a catalytic core of four LSU dimers, capped by four SSUs on both sides of C helix B a solvent channel (Fig. 1B). Based on the crystal structures (22, 23), and internal regions affecting Rubisco active site (24), we focused on the two solvent-exposed α-helices of the Rubisco helix A SSU, to determine whether differences in Rubisco packaging between pyrenoidless higher plant hybrid enzymes, as repre- sented by the spinach hybrid (21), and wild-type Chlamydomo- nas, could be mediated by extrinsic protein interactions. The two α-helices (A and B) contribute to one-third of the SSU surface- exposed residues (Fig. 1B). The spinach and Chlamydomonas D 23 35 86 99 SSUs are structurally very similar (Fig. 1C), and the two α-helices | | | | have an identical number of residues (24) but differ markedly in Cr DEQIAAQVDYIVA PMQVLREIVACTKA amino acid composition (Fig. 1D). Transformation vectors con- So TDQLARQVDYLLN PAQVLNELEECKKE taining cDNA encoding for either spinach or Chlamydomonas SSU (21) were