title: Ribulose-1,5-bis-phosphate carboxylase/oxygenase accumulation factor1 is required for holoenzyme assembly in maize.
abstract: Most life is ultimately sustained by photosynthesis and its rate-limiting carbon fixing enzyme, ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). Although the structurally comparable cyanobacterial Rubisco 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 Rubisco 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 Rubisco deficient and seedling lethal. Analysis of transcripts and proteins showed that Rubisco large subunit synthesis in raf1 plants is not compromised; however, newly synthesized Rubisco 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 Rubisco large subunit, suggesting that they interact weakly or transiently. RAF1 is predominantly expressed in bundle sheath chloroplasts, consistent with a Rubisco accumulation function. Our results support the hypothesis that RAF1 acts during Rubisco assembly by releasing and/or sequestering the large subunit from chaperonins early in the assembly process.
authors.affiliation: Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Leila
authors.initials: L
authors.lastname: Feiz
authors.forename: Rosalind
authors.initials: R
authors.lastname: Williams-Carrier
authors.forename: Katia
authors.initials: K
authors.lastname: Wostrikoff
authors.forename: Susan
authors.initials: S
authors.lastname: Belcher
authors.forename: Alice
authors.initials: A
authors.lastname: Barkan
authors.forename: David B
authors.initials: DB
authors.lastname: Stern
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D060365:Chloroplast Proteins / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology / Q000235:genetics; D032461:Chromosomes, Plant / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005816:Genetic Complementation Test; D020035:Holoenzymes / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
chemical_list: D060365:Chloroplast Proteins; D004251:DNA Transposable Elements; D020035:Holoenzymes; D018832:Molecular Chaperones; D010940:Plant Proteins; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase