MalFGK complex assembly and transport and regulatory characteristics of MalK insertion mutants

MalK is a peripheral cytoplasmic membrane protein that has multiple activities in Escherichia coli. It associates with integral cytoplasmic membrane proteins MalF and MalG to form the maltose transport complex (MalFGK), a member of the ATP-binding cassette (ABC) superfamily of proteins, In addition, MalK participates in two different regulatory pathways which modulate mal gene expression and MalFGK transport activity, We have created a set of malK mutations for analysis of the protein's structure and folding, These mutations, distributed throughout malK, are all similar insertions of 31 codons. The ability of each mutant to function in maltose transport and MalK-dependent regulation was characterized, Furthermore, we have exploited a sensitive biochemical assay to classify our MalK insertion mutants into two additional categories: MalFGK complex assembly proficient and complex assembly defective, The regions containing the insertions in the assembly-proficient class should correspond to areas within MalK that are surface exposed within the MalFGK complex, Affected regions in assembly-deficient mutants may be involved in critical structural contacts within the complex, One mutant apparently blocks assembly at an intermediate stage prior to oligomerization of the final MalFGK complex, This work contributes to the analysis of ABC transport proteins and to the study of the assembly process for hetero-oligomeric membrane proteins.