title: Isolation and compositional analysis of a CP12-associated complex of calvin cycle enzymes from Nicotiana tabacum.
abstract: Two Calvin Cycle enzymes, NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a multiprotein complex with CP12, a small intrinsically-unstructured protein. Under oxidizing conditions, association with CP12 confers redox-sensitivity to the otherwise redox-insensitive A isoform of GAPDH (GapA) and provides an additional level of down-regulation to the redox-regulated PRK. To determine if CP12-mediated regulation is specific for GAPDH and PRK in vivo, a high molecular weight complex containing CP12 was isolated from tobacco chloroplasts and leaves and its protein composition was characterized. Gel electrophoresis and immunoblot analyses after separation of stromal proteins by size fractionation verified that the GAPDH (both isoforms) and PRK co-migrated with CP12 in dark- but not light-adapted chloroplasts. Nano-liquid-chromatography-mass-spectrometry of the isolated complex identified only CP12, GAPDH and PRK. Since nearly all of the CP12 from darkened chloroplasts migrates with GADPH and PRK as a high molecular mass species, these data indicate that the tight association of tobacco CP12 with GAPDH and PRK is specific and involves no other Calvin Cycle enzymes.
authors.affiliation: Arid-Land Agricultural Research Center, Agricultural Research Service, US Department of Agriculture, 21881 N. Cardon Lane, Maricopa, Arizona 85138, USA.
authors.forename: A Elizabete
authors.initials: AE
authors.lastname: Carmo-Silva
authors.forename: Lucia
authors.initials: L
authors.lastname: Marri
authors.forename: Francesca
authors.initials: F
authors.lastname: Sparla
authors.forename: Michael E
authors.initials: ME
authors.lastname: Salvucci
mesh_terms: D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000378:metabolism; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D010788:Photosynthesis; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase