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 example 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 translocate maltose and maltodextrins across the bacterial cytoplasmic membrane. To investigate the role of MalG both in MalFGK2 assembly interactions and in subsequent transport interactions, we isolated and characterized 18 different MalG mutants, each containing a 31-residue insertion in the protein. Eight insertions mapping to distinct hydrophilic regions of MalG permitted either assembly or both assembly and transport interactions to occur. In particular, we isolated two insertions mapping to extracytoplasmic (periplasmic) regions of MalG which preserved both assembly and transport abilities, suggesting that these are permissive sites in the protein. Another periplasmic insertion seems to affect only transport-specific interactions between MalG and maltose-binding protein, defining a novel class of MalG mutants. Finally, four MalG mutant proteins, although stably expressed, are unable to assemble into the MalFGK2 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 assembly determinants. These MalG mutants will allow us to further explore the intermolecular interactions of this model transporter.
Nelson
B D
BD
Department of Microbiology, University of Washington, Seattle 98195-7242, USA.
Traxler
B
B
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005799:Genes, Dominant; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016254:Mutagenesis, Insertional; D033902:Periplasmic Binding Proteins; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; C079123:MalG protein, E coli; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C461599:maltose transport system, E coli; D008320:Maltose