A bacterial system for investigating transport effects of cystic fibrosis--associated mutations.
LIV-I, a high-affinity system that transports neutral, branched-chain amino acids into Escherichia coli, has two components, LivG and LivF, that are homologous to the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). CF-associated mutations of human CFTR were introduced into corresponding regions of LivG, and their effects on leucine transport could be grouped into three classes. Mutations were found that (i) abolished LIV-I--directed transport, (ii) retained about a quarter of wild-type activity at the Michaelis-Menten constant (KM), and (iii) had minimal activity at the KM. A mutation equivalent to a benign polymorphism had no effect on transport. The correlation of these mutational phenotypes in LivG and CFTR suggests that the LIV-I prokaryotic transporter is functionally similar to the CF protein and that this similarity can be exploited to clarify the properties of the nucleotide-binding fold in this superfamily of proteins.
Gibson
A L
AL
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Wagner
L M
LM
Collins
F S
FS
Oxender
D L
DL
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D015183:Restriction Mapping; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C505032:CFTR protein, human; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C064913:livF protein, E coli; C064912:livG protein, E coli; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D007930:Leucine