In vivo detection of hetero-association of glycophorin-A and its mutants within the membrane.
Protein recognition within the membrane is a crucial process for numerous biological activities. Detection of such interaction is limited because of difficulties that arise from the hydrophobic environment of the membrane. We detected direct hetero-oligomerization of the glycophorin-A (GPA) transmembrane segments in vivo through inhibition of ToxR transcription activator dimer formation. We investigated the amino acids important for hetero-oligomerization within the membrane, using peptide analog segments of the transmembrane domain of glycophorin A. The wild type ([WT]GPA) and alanine mutant ([A]GPA) were able to interfere with and inhibit the proper dimerization of the ToxR-GPA transcription factor. Conversely, a second alanine mutant ([A(2)]GPA), a glycine mutant ([G]GPA), and a scrambled analog ([SC]GPA) were virtually inactive. Binding studies reveal similar membrane partitions for [WT]GPA, [G]GPA, and [SC]GPA, whereas membrane partition of [A]GPA and [A(2)]GPA are lower. Spectral analysis of fluorescent-labeled analogs revealed a significant blue shift, indicating membrane insertion. Our results suggest that the GXXXG motif, found in homo-oligomerization, is not sufficient for hetero-oligomerization in a biological membrane, whereas an extended motif, LIXXGXXXGXXXT, is sufficient. Interfering with hetero-oligomerization within the membrane can be a useful strategy for characterizing such interactions and possibly modulating membrane protein activity.
Gerber
D
D
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel.
Shai
Y
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D006021:Glycophorins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D014157:Transcription Factors / Q000737:chemistry
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D006021:Glycophorins; D014157:Transcription Factors; C066719:toxR protein, bacteria