Ribulose-15-Bis-Phosphate Carboxylase/Oxygenase Accumulation Factor1 Is Required for Holoenzyme Assembly in Maize

摘要

Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (). Although the structurally comparable cyanobacterial is amenable to in vitro assembly, the higher plant enzyme has been refractory to such manipulation due to poor understanding of its assembly pathway. Here, we report the identification of a chloroplast protein required for accumulation in maize (Zea mays), RUBISCO ACCUMULATION FACTOR1 (RAF1), which lacks any characterized functional domains. Maize lines lacking RAF1 due to Mutator transposon insertions are deficient and seedling lethal. Analysis of transcripts and proteins showed that large subunit synthesis in raf1 plants is not compromised; however, newly synthesized large subunit appears in a high molecular weight form whose accumulation requires a specific chaperonin 60 isoform. Gel filtration analysis and blue native gels showed that endogenous and recombinant RAF1 are trimeric; however, following in vivo cross-linking, RAF1 copurifies with large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a accumulation function. Our results support the hypothesis that RAF1 acts during assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process.