Structure-function study of MalF protein by random mutagenesis

MalF is one of the tno integral inner membrane proteins of the maltose-malt odextrin transport system. To identify functional regions in this protein, we characterized a collection of malF mutants obtained by random mutagenesi s. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcell ular localization of MalK, Only 2 of the 21 MalF mutant proteins allowed gr owth on maltose and maltodextrins. Most mutations resulting in immunodetect able proteins mapped to hydrophilic domains, indicating that insertions aff ecting transmembrane segments gave rise to unstable or lethal proteins, All MalF mutant proteins, even those C-terminally truncated or with large N-te rminal deletions, were inserted into the cytoplasmic membrane. Having ident ified mutations leading to reduced steady-state level, to partial mislocati on, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK(2), complex and/or in the transport mech anism.