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 b iological activities. Detection of such interaction is limited because of d ifficulties that arise from the hydrophobic environment of the membrane. We detected direct hetero-oligomerization of the glycophorin-A (GPA) transmem brane segments in vivo through inhibition of ToxR transcription activator d imer formation. We investigated the amino acids important for hetero-oligom erization within the membrane, using peptide analog segments of the transme mbrane 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]G PA are lower. Spectral analysis of fluorescent-labeled analogs revealed a s ignificant blue shift, indicating membrane insertion. Our results suggest t hat the GXXXG motif, found in homo-oligomerization, is not sufficient for h etero-oligomerization in a biological membrane, whereas an extended motif, LIXXGXXXGXXXT, is sufficient. Interfering with hetero-oligomerization withi n the membrane can be a useful strategy for characterizing such interaction s and possibly modulating membrane protein activity.