EXPLORING THE ROLE OF INTEGRAL MEMBRANE-PROTEINS IN ATP-BINDING CASSETTE TRANSPORTERS - ANALYSIS OF A COLLECTION OF MALG INSERTION MUTANTS

The maltose transport complex of Escherichia coli is a well-studied ex ample of an ATP-binding cassette transporter, The complex, containing one copy each of the integral membrane proteins MalG and MalF and two copies of the peripheral cytoplasmic membrane protein MalK, interacts with the periplasmic maltose-binding protein to efficiently translocat e maltose and maltodextrins across the bacterial cytoplasmic membrane. To investigate the role of MalG both in MalFGK(2) assembly interactio ns and in subsequent transport interactions, we isolated and character ized 18 different MalG mutants, each containing a 31-residue insertion in the protein. Eight insertions mapping to distinct hydrophilic regi ons of MalG permitted either assembly or both assembly and transport i nteractions to occur. In particular, we isolated two insertions mappin g to extracytoplasmic (periplasmic) regions of MalG which preserved bo th assembly and transport abilities, suggesting that these are permiss ive sites in the protein. Another periplasmic insertion seems to affec t only transport-specific interactions between MalG and maltose-bindin g protein, defining a novel class of MalG mutants. Finally, four MalG mutant proteins, although stably expressed, are unable to assemble int o the MalFGK(2) complex. These mutants contain insertions in only two different hydrophilic regions of MalG, consistent with the notion that a restricted number of domains in this protein are critical complex a ssembly determinants. These MalG mutants will allow us to further expl ore the intermolecular interactions of this model transporter.