Membrane proteins from the cyanobacterium Synechocystis sp. PCC 6803 interacting with thioredoxin.
Cysteine dithiol/disulphide exchange forms the molecular basis for regulation of a wide variety of enzymatic activities and for transduction of cellular signals. Thus, the search for proteins with reactive, accessible cysteines is expected to contribute to the unravelling of new molecular mechanisms for enzyme regulation and signal transduction. Several methods have been designed for this purpose taking advantage of the interactions between thioredoxins and their protein substrates. Thioredoxins comprise a family of redox-active enzymes, which catalyse reduction of protein disulphides and sulphenic acids. Due to the inherent practical difficulties associated with studies of membrane proteins these have been largely overlooked in the many proteomic studies of thioredoxin-interacting proteins. In the present work, we have developed a procedure to isolate membrane proteins interacting with thioredoxin by binding in situ to a monocysteinic His-tagged thioredoxin added directly to the intact membranes. Following fractionation and solubilisation of the membranes, thioredoxin target proteins were isolated by Ni-affinity chromatography and 2-DE SDS-PAGE under nonreducing/reducing conditions. Applying this method to total membranes, including thylakoid and plasma membranes, from the cyanobacterium Synechocystis sp. PCC 6803 we have identified 50 thioredoxin-interacting proteins. Among the 38 newly identified thioredoxin targets are the ATP-binding subunits of several transporters and members of the AAA-family of ATPases.
Mata-Cabana
Alejandro
A
Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas - Universidad de Sevilla, Sevilla, Spain.
Florencio
Francisco J
FJ
Lindahl
Marika
M
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D025941:Protein Interaction Mapping; D040901:Proteomics; D046939:Synechocystis / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D013879:Thioredoxins