Regulation of flavin dehydrogenase compartmentalization: requirements for PutA-membrane association in Salmonella...

PutA is a multifunctional, peripheral membrane protein which functions both as an autogenous transcriptional repressor and the enzyme which catalyzes the two-step conversion of proline to glutamate in Salmonella typhimurium and Escherichia coli. To understand how PutA associates with the membrane, we determined the role of FAD redox and membrane components in PutA-membrane association. Reduction of the tightly bound FAD is required for both derepression of the put operon and membrane association of PutA. FADH(2) alters the conformation of PutA, resulting in an increased hydrophobicity. Previous studies used enzymatic activity as an assay for membrane association and concluded that electron transfer from the reduced FAD in PutA to the membrane is required for the PutA-membrane interaction. However, direct physical assays of PutA association with membrane vesicles from quinone deficient mutants demonstrated that although electron transfer is essential for proline dehydrogenase activity, it is not required for PutA-membrane association per se. Furthermore, PutA efficiently associated with liposomes, indicating that PutA-membrane association does not require interactions with other membrane proteins. PutA enzymatic activity can be efficiently reconstituted with liposomes containing ubiquinone and cytochrome bo, confirming that proline dehydrogenase can pass electrons directly to the quinone pool. These results indicate that PutA-membrane association is due strictly to a protein-lipid interaction initiated by reduction of FAD.