Analysis of Carboxysomes from Synechococcus PCC7942 Reveals Multiple Rubisco Complexes with Carboxysomal Proteins CcmM and CcaA

Abstract

In cyanobacteria, the key enzyme for photosynthetic CO 2 fixation, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is bound within proteinaceous polyhedral microcompartments called carboxysomes. Cyanobacteria with Form IB Rubisco produce ␤-carboxysomes whose putative shell proteins are encoded by the ccm-type genes. To date, very little is known of the protein-protein interactions that form the basis of ␤-carboxysome structure. In an effort to identify such interactions within the carboxysomes of the ␤-cyanobacterium Synechococcus sp. PCC7942, we have used polyhistidine-tagging approaches to identify at least three carboxysomal subcomplexes that contain active Rubisco. In addition to the expected L 8 S 8 Rubisco, which is the major component of carboxysomes, we have identified two Rubisco complexes containing the putative shell protein CcmM, one of which also contains the carboxysomal carbonic anhydrase, CcaA. The complex containing CcaA consists of Rubisco and the full-length 58-kDa form of CcmM (M58), whereas the other is made up of Rubisco and a short 35-kDa form of CcmM (M35), which is probably translated independently of M58 via an internal ribosomal entry site within the ccmM gene. We also show that the high CO 2 -requiring ccmM deletion mutant (⌬ccmM) can achieve nearly normal growth rates at ambient CO 2 after complementation with both wild type and chimeric (His 6 -tagged) forms of CcmM. Although a significant amount of independent L 8 S 8 Rubisco is confined to the center of the carboxysome, we speculate that the CcmMCcaA-Rubisco complex forms an important assembly coordination within the carboxysome shell. A speculative carboxysome structural model is presented.Prokaryotic organisms are distinct from their eukaryotic counterparts by the absence of specialized membrane-bound compartments. Although it is commonly considered that prokaryotic cells do not compartmentalize cellular processes, there is growing evidence that cyanobacteria and some ␤-proteobacteria have the means to do so when certain enzymes need to be sequestered or protected for maximal catalytic activity (1-3). Carboxysomes, for example, are polyhedral protein microcompartments found in cyanobacteria and chemoautotrophic bacteria, which contain the majority of a cell's ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (4). These microcompartments are essential to the operation of the CO 2 -concentrating mechanism in cyanobacteria. The CO 2 -concentrating mechanism utilizes a number of inorganic carbon (C i , the sum of CO 2 and HCO 3 Ϫ ) transport systems to elevate cellular HCO 3 Ϫ concentrations prior to CO 2 fixation (for reviews, see Refs. 5 and 6). Rubisco is a hexadecameric enzyme with a core of eight large subunits (L 8 ) made up of four L 2 dimers, each containing two catalytic sites. Attached to each end of the L 8 core are four small subunits (S 8 ) that are necessary for catalytic competence (7). Rubisco carries out the CO 2 fixation process in the initial step of the Calvin cycle, yet it is characterized by ...

Genetic diversity of inorganic carbon uptake systems causes variation in CO2 response of the cyanobacterium Microcystis

“…Only 4 of our 20 strains contained both ccaA and ccaA2. The amino-acid sequence of CcaA2 is slightly shorter than CcaA, and the two different isoforms might enable cells to modify their CO 2 fixation efficiency (Long et al, 2007), although further confirmation of this hypothesis is warranted.…”

Insights from multiple structures of the shell proteins from the β‐carboxysome

“…32 How the interior of the carboxysome is organized relative to the shell is understood only in rough terms. 23,24 Two recent studies have highlighted the role of the CcmM protein in the b-type carboxysome in binding several other carboxysome proteins, 28,29 including the CcmK shell proteins. 29 The structural details of these interactions have not been illuminated yet.…”

“…Two independent studies on b-caboxysomes suggest that the CcmM protein plays a key organizing role. 28,29 In one study, 29 yeast two hybrid experiments showed direct interactions between CcmM and the CcmK (BMC-type) shell proteins under study in the present work. The atomic level details of those interactions have not been elucidated yet.…”

Modularity of a carbon-fixing protein organelle

“…Its shell is assembled from roughly 800 protein hexamers, which form the facets of the icosahedron, and 12 pentamers, which form the vertices (8,12). Inside the lumen are the enzymes ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase (CA), which are directed to this location by protein-protein interactions with the shell and/or shell interaction mediators (13). Depending on the organism, genes for these shell proteins and luminal enzymes can occur either together in a single operon or, alternatively, in several operons across the genome (9).…”