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

Abstract

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.Maltose transport across the cytoplasmic membrane of Escherichia coli utilizes a periplasmic binding protein-dependent transporter which is a member of the ATP-binding cassette (ABC or traffic ATPase) superfamily of proteins (reviewed in references 2, 8, and 14). In addition to the prokaryotic binding protein-dependent transporters, the ABC superfamily also includes several medically important eukaryotic proteins, including mammalian P-glycoprotein of multidrug-resistant tumor cells and the cystic fibrosis transmembrane conductance regulator. The maltose permease is a heterotetrameric complex comprised of integral membrane proteins MalF and MalG, which associate with peripheral membrane protein MalK in the stochiometry MalFGK 2 (4). Our goal is to characterize the assembly and folding of the MalFGK 2 complex to better understand the process of tertiary and quaternary folding of membrane proteins and the structural and functional interactions within the complex. Knowledge gained by the study of maltose permease also should contribute to the structure-function analysis of the other ABC superfamily proteins.Here, we describe the results of our mutagenesis of the gene coding for MalK, which is the domain of the maltose transporter with the most extensive homology to other ABC transporters. MalK has been shown to have several distinct functions in the cell. First, MalK contains a well-defined nucleotide-binding fold (Walker boxes A and B) that has been shown to be essential for the hydrolysis of ATP that energizes maltose transport (4,6,9,23). Second, MalK contributes to the regulation of mal gen...

The Crystal Structure of the MJ0796 ATP-binding Cassette

“…Furthermore, there are many features of the Rad50 dimer that are consistent with previous biochemical experimentation on ABC transporter proteins (35)(36)(37)(38)(39) (Figs. 4 and 5).…”

Novel Missense Mutations That Affect the Transport Function of MalK, the ATP-Binding-Cassette Subunit of the Salmonella enterica Serovar Typhimurium Maltose Transport System

“…The mechanism of this activity is currently unknown, but evidence for binding of MalK to MalT, the positive regulator of the maltose regulon, has recently been presented (12). These and other functions can be separated by mutations (8,9); this finding, together with data from the study of half molecules and chimeras (13), has led to the proposal of a domain structure as presented in Fig. 1.…”

Evidence for Multiple Pathways in the Assembly of the Escherichia coli Maltose Transport Complex

“…When co-expressed, malF and malG alleles were carried by the same plasmid. Insertion mutations in malK and malG were described previously (17,20). malFi178 and malFi410 contain a 31-codon insertion (i31) in codons 178 and 410, respectively, of the malF gene.…”

“…In that study, the Mal proteins were expressed from the chromosomal genes and were immunoprecipitated from steady-state labeled membrane preparations to look specifically at the stoichiometry of the fully assembled complex. The relative proportions of Mal sub- (19) and MalK550, the i31 mutant after Gln-5 (20). units in our immunoprecipitations likely are because of our conditions, which are designed to examine the assembly process and to facilitate the characterization of mutants.…”