Proteomics

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FLAG-affinity pulldown experiments with sll1214 and sll1874 from Synechocystis

ABSTRACT: Putative subunits of the Mg-protoporphyrin monomethylester oxidative cyclase from Synechocystis PCC 6803, sll1214 and sll1874 were expressed with N-terminal 3xFLAG-tags in separate transformants.. Solubilised thylakoid membranes from these 2 transformants were incubated with anti-FLAG agarose and after extensive washing, the FLAG-tagged proteins, together with putative interaction partners were desorbed with FLAG peptide. The eluates were denatured, reduced, alkylated and digested with trypsin. The resulting peptides were cleaned by cation exchange and desalted, then analysed by nanoLC-MS/MS. The FLAG pulldown experiment was also carried out with a thylakoid membrane preparation from wild-type with no FLAG-tagged proteins as a control. Protein identification was by database searching with Mascot.

INSTRUMENT(S): instrument model

ORGANISM(S): Synechocystis Sp. (strain Pcc 6803 / Kazusa)

SUBMITTER: Philip Jackson  

PROVIDER: PXD000632 | Pride | 2015-08-05

REPOSITORIES: Pride

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Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803.

Hollingshead Sarah S   Kopecná Jana J   Jackson Philip J PJ   Canniffe Daniel P DP   Davison Paul A PA   Dickman Mark J MJ   Sobotka Roman R   Hunter C Neil CN  

The Journal of biological chemistry 20120618 33

The cyclase step in chlorophyll (Chl) biosynthesis has not been characterized biochemically, although there are some plausible candidates for cyclase subunits. Two of these, Sll1214 and Sll1874 from the cyanobacterium Synechocystis 6803, were FLAG-tagged in vivo and used as bait in separate pulldown experiments. Mass spectrometry identified Ycf54 as an interaction partner in each case, and this interaction was confirmed by a reciprocal pulldown using FLAG-tagged Ycf54 as bait. Inactivation of th  ...[more]

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