Compartmentalization of enzymes is a cellular strategy to regulate metabolic pathways and increase their efficiency 1 . The αand β-carboxysomes of cyanobacteria contain Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a complex of 8 large (RbcL) and 8 small (RbcS) subunits, and carbonic anhydrase (CA) 2-4 . Since the proteinaceous carboxysome shell provides a barrier to the diffusion of CO2 but not HCO3 − (ref. 5), CA generates high concentrations of CO2 for carbon fixation by Rubisco 6 . The shell also prevents access to reducing agents, generating an oxidizing environment 7-9 . Formation of β-carboxysomes involves aggregation of Rubisco by the protein CcmM 10 , which exists in two forms: Fulllength CcmM (M58 in Synechococcus elongatus PCC7942) containing a CA-like domain 8 followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers, and M35, lacking the CA-like domain 11 . It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS 2-4 . Here we reconstituted the Rubisco:CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for carboxysome function in vivo. Importantly, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low complexity sequences, which typically mediate liquid-liquid phase separation in eukaryotes 12,13 . Indeed, within the pyrenoid of eukaryotic algae, the functional homologue of carboxysomes, Rubisco has been shown to adopt a liquid-like state via interactions with the intrinsically disordered protein EPYC1 14 . Understanding carboxysome biogenesis will be important in efforts to engineer CO2 concentrating mechanisms (CCM) in plants 15-19 .
Plant RuBisCo, a complex of eight large and eight small subunits, catalyzes the fixation of CO in photosynthesis. The low catalytic efficiency of RuBisCo provides strong motivation to reengineer the enzyme with the goal of increasing crop yields. However, genetic manipulation has been hampered by the failure to express plant RuBisCo in a bacterial host. We achieved the functional expression of RuBisCo in by coexpressing multiple chloroplast chaperones. These include the chaperonins Cpn60/Cpn20, RuBisCo accumulation factors 1 and 2, RbcX, and bundle-sheath defective-2 (BSD2). Our structural and functional analysis revealed the role of BSD2 in stabilizing an end-state assembly intermediate of eight RuBisCo large subunits until the small subunits become available. The ability to produce plant RuBisCo recombinantly will facilitate efforts to improve the enzyme through mutagenesis.
FtsHs are a well-characterized family of membrane bound proteases containing an AAA (ATPase associated with various cellular activities) and a Zn(2+) metalloprotease domain. FtsH proteases are found in eubacteria, animals and plants and are known to have a crucial role in housekeeping proteolysis of membrane proteins. In Arabidopsis thaliana, 12 FtsH family members are present (FtsH 1-12) and their subcellular localization is restricted to mitochondria and chloroplasts. In addition, five genes coding for proteins homologous to FtsH (FtsHi 1-5) have been detected in the genome, lacking the conserved zinc-binding motif HEXXH, which presumably renders them inactive for proteolysis. These inactive FtsHs as well as nine of the active FtsHs are thought to be localized in the chloroplast. In this article, we shortly summarize the recent findings on plastidic FtsH proteases in text and figures. We will mainly focus on FtsH 1, 2, 5 and 8 localized in the thylakoid membrane and known for their importance in photosynthesis.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the fixation of CO2 in photosynthesis. Despite its pivotal role, Rubisco is an inefficient enzyme and thus is a key target for directed evolution. Rubisco biogenesis depends on auxiliary factors, including the GroEL/ES-type chaperonin for folding and the chaperone RbcX for assembly. Here we performed directed evolution of cyanobacterial form I Rubisco using a Rubisco-dependent Escherichia coli strain. Overexpression of GroEL/ES enhanced Rubisco solubility and tended to expand the range of permissible mutations. In contrast, the specific assembly chaperone RbcX had a negative effect on evolvability by preventing a subset of mutants from forming holoenzyme. Mutation F140I in the large Rubisco subunit, isolated in the absence of RbcX, increased carboxylation efficiency approximately threefold without reducing CO2 specificity. The F140I mutant resulted in a ∼55% improved photosynthesis rate in Synechocystis PCC6803. The requirement of specific biogenesis factors downstream of chaperonin may have retarded the natural evolution of Rubisco.
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