Insight into Ribulose 1,5-Bis-Phosphate Carboxylase/Oxygenase Assembly in Maize

Widely heralded as the most important enzyme on Earth, ribulose 1,5-bis-phosphate carboxylase/oxygenase (Rubisco) catalyzes the reaction that draws inorganic carbon into the biosphere during the Calvin-Benson cycle. In higher plants, algae, and cyanobacteria, Rubisco occurs as a hexadecamer consisting of eight large (50 kD) subunits and eight small (13 to 15 kD) subunits. Whereas the large subunits are encoded in the chloroplast genome in plants, the small subunits are nuclear gene products that travel as preproteins to the chloroplast, where they lose their signal peptides before combining with large subunits to form a stable holoenzyme. Attempts to express higher plant Rubisco in bacteria have failed, suggesting that additional, plant-specific factors are required for Rubisco biogenesis.

In a quest to identify components involved in Rubisco assembly in maize (Zea mays), Feiz et al. (pages 3435–3446) screened the Photosynthetic Mutant Library, a collection of Mu transposon–induced nonphotosynthetic maize mutants (Stern et al., 2004), for lineages with reduced levels of Rubisco. The raf1-1 mutant lacked Rubisco and perished within 1 month of germination. Mu insertions in the GRMZM2G457621 locus were found to underlie the mutant phenotype. Consistent with its expected involvement in photosynthesis, Rubisco Accumulation Factor1 (RAF1) was predicted to contain an N-terminal chloroplast targeting peptide, and transcriptome analysis done independently revealed that, like RNAs encoding Rubisco subunits, RAF1 mRNA was enriched in bundle sheath cells (Li et al., 2010).

RNA and protein analyses revealed that Rubisco subunit genes underwent normal transcription and translation in raf1 plants, suggesting that RAF1 contributes to Rubisco biosynthesis by mediating its assembly or stabilizing the holoenzyme. In the raf1 mutant, the Rubisco large subunit was found to accumulate as part of a large complex rather than as part of a holoenzyme. Using liquid chromatography–tandem mass spectrometry, the authors identified Cpn60, a chaperonin protein known to promote Rubisco assembly and activity (Goloubinoff et al., 1989), as a member of this complex. The authors proposed that RAF1 functions at a postchaperonin step to fold and/or assemble the Rubisco large subunit into the holoenzyme.

An analysis of endogenous and recombinant RAF1 using blue native polyacrylamide gel electrophoresis demonstrated that RAF1 occurs as a trimer. The authors demonstrated that RAF1 interacts directly with the Rubisco large subunit, and that this interaction is strengthened when RAF1 is stabilized by cross-linking (see figure). RAF1 therefore likely functions as an assembly chaperone by releasing and/or sequestering the Rubisco large subunit from chaperonins during holoenzyme biogenesis.

RAF1 interacts directly with the Rubisco large subunit and RAF1 crosslinking strengthens this interaction. Immunoblots were probed with Rubisco LS or RAF1 antibodies. Samples were proteins isolated from maize leaves (input), proteins precipitated with RAF1 antibody without or following (XL) crosslinking, and a control using an unrelated antibody and crosslinked proteins. (Reprinted and adapted from Feiz et al. [2012], Figure 6.)

This study represents an important advance in understanding higher plant Rubisco assembly.