Structure-function study of MalF protein by random mutagenesis.
MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system. To identify functional regions in this protein, we characterized a collection of malF mutants obtained by random mutagenesis. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK. Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane. Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK2 complex and/or in the transport mechanism.
Tapia
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Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS URA 1444, Institut Pasteur, F75724 Paris Cedex 15, France.
Mourez
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Hofnung
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Dassa
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D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; C068021:MalK protein, Bacteria; D058619:Maltose-Binding Proteins