The state of oligomerization of Rubisco controls the rate of synthesis of the Rubisco large subunit in Chlamydomonas reinhardtii

Abstract

Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is present in all photosynthetic organisms and is a key enzyme for photosynthesis-driven life on Earth. Its most prominent form is a hetero-oligomer in which small subunits (SSU) stabilize the core of the enzyme built from large subunits (LSU), yielding, after a chaperone-assisted multistep assembly process, an LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii and a combination of site-directed mutants to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU are associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis, in which LSU translation is controlled by its ability to assemble with the SSU, via the mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model whereby the last assembly intermediate, an LSU8-RAF1 complex, provides the platform for SSU binding to form the Rubisco enzyme, and when SSU is not available, converts to a key regulatory form that exerts negative feed-back on the initiation of LSU translation.

Transit Peptides Often Require Downstream Unstructured Sequence for Efficient Chloroplast Import in Chlamydomonas reinhardtii

“…Constructs were Gibson assembled as described in Garrido et al (2020) based on pMO611, a variant of published pMO449 ( Onishi and Pringle, 2016 ) where an upstream start codon was mutated to CTG. Sequences inserted upstream of Venus were amplified from Chlamydomonas genomic DNA extracted from wild-type strain T222+, with the following exceptions: construct “RBCS2-cTP + 23” was amplified from cDNA generated from T222+ to avoid introducing an intron not present in the other RBCS2-cTP constructs; the G-rich artificial linker sequence was amplified from plasmid CrRaf1-pJHL encoding Raf1-strep ( Wietrzynski et al, 2021 ). Corresponding amino acid sequences are given in Table 1 .…”

Retrograde and anterograde signaling in the crosstalk between chloroplast and nucleus

“…Folding of newly synthesized LSU is mediated by the Cpn60/Cpn20 chaperonin complex. This is followed by formation of LSU oligomers consisting of four dimers and by the assembly of the holoenzyme L 8 S 8 , a process which is assisted by the chaperones RbcX, Raf1, and Raf2 (Aigner et al, 2017;Wietrzynski et al, 2021). In both land plants and Chlamydomonas, the stoichiometric expression of the two Rubisco subunits is regulated by the CES process (control by epistasy of synthesis) that represses the translation of LSU in the absence of SSU (Wostrikoff and Stern, 2007).…”

Chloroplast transit peptides often require downstream unstructured sequence in Chlamydomonas reinhardtii

“…Constructs were Gibson assembled as described in (Garrido et al, 2020) based on pMO611, a variant of published pMO449 (Onishi and Pringle, 2016) where an upstream start codon was mutated to CTG. Sequences inserted upstream of Venus were amplified from Chlamydomonas genomic DNA extracted from wild-type strain T222+, with the following exceptions: construct ‘RBCS-cTP+23’ was amplified from cDNA generated from T222+ to avoid introducing an intron not present in the other RBCS-cTP constructs; the G-rich artificial linker sequence was amplified from plasmid CrRaf1-pJHL encoding Raf1-strep (Wietrzynski et al, 2021). Corresponding amino acid sequences are given in Table 1.…”