Structural arrangement of lens fiber cell plasma membrane protein MP20

The membrane topology of the bovine lens fiber cell plasma membrane protein MP20 has been examined using anti-peptide antibodies and the hydrophobic label 3-(trifluoromethyl)-3-(m-[I-125]iodophenyl)diazerine ([I-125]TID). The specificity of the affinity-purified anti-peptide polyclonal antibodies, directed against four separate hydrophilic segments of MP20, was established by immunodot blots, Western immunoblotting and ELISA. Western immunodetection of protease-treated, urea-washed lens membranes indicated that each of the segments of MP20 identified by the anti-peptide antibodies was accessible to proteases indicating their likely extramembranous location. Immunoelectron microscopy of junctional lens membrane immunolabeled with MP20 anti-peptide antibodies directed against two segments predicted to be on the extra-cellular face of the lens fiber cell plasma membrane suggests these segments may actually be located on the cytoplasmic plasma membrane face. Transmembrane segments of MP20, identified using the hydrophotic photo-affinity label [I-125]TID, were isolated and sequenced. Only three of the four previously proposed transmembrane segments of this protein were significantly labeled with this reagent. Based on these results and previously reported information regarding MP20, a new topological model. is proposed for the arrangement of MP20 in the lens fiber cell plasma membrane. The new topological model of MP20 includes two alpha-helical and two beta-strand transmembrane segments. (C) 1998 Academic Press Limited.