CHARACTERIZATION OF TRANSMEMBRANE DOMAIN-6, DOMAIN-7, AND DOMAIN-8 OFMALF BY MUTATIONAL ANALYSIS

Oligonucleotide mutagenesis was used to isolate mutations in membrane- spanning segments 6, 7, and 8 of MalF, MalF is a cytoplasmic membrane component of the binding protein-dependent maltose transport system in Escherichia coli. The current structural model predicts eight transme mbrane domains for MalF, Membrane-spanning segments 6, 7, and 8 of Mal F flank or are part of the EAA-X(3)-G-X(9)-I-X-LP consensus region pre sent in the cytoplasmic membrane subunits of the bacterial ABC transpo rter superfamily members, Mutations with two novel phenotypes with res pect to substrate specificity of the maltose transport system were iso lated, One mutant grew on minimal maltose media but not on media conta ining either maltoheptaose or maltoheptaose plus maltose and was thus termed dextrin dominant negative. The other class of mutations led to a maltose minus but maltoheptaose plus phenotype. Nine of the isolated mutations leading to changes in substrate specificity were tightly cl ustered on one face of the postulated transmembrane helix 6. A similar clustering of mutations was detected in transmembrane domain 7. The m ajority of mutations in membrane-spanning segment 7 led to a protease- sensitive or a conditional phenotype with respect to MalF function or both. Mutations in transmembrane domain 8 appeared to be more randomly distributed, The majority of mutations in membrane-spanning segment 8 caused a Mal(+) Dex(-) phenotype, Six Mal(+) suppressor mutations iso lated to two mutations in transmembrane domain 7 changed amino acid re sidues in membrane-spanning segment 6 or 8.