CYTOSOLIC FACTORS BLOCK ANTIBODY-BINDING TO THE C-TERMINAL CYTOPLASMIC TAIL OF THE KDEL RECEPTOR

The mammalian KDEL receptor is an extremely hydrophobic membrane prote in. One of the longest stretches of hydrophilic sequence resides at th e C terminus. Various antibodies against a synthetic peptide correspon ding to this region confirmed that the C-terminus is exposed to the cy toplasm. It was observed that antibody binding to the C-terminus of th e KDEL receptor was diminished during immunofluorescence microscopy pr ocedures which involved fixation prior to permeabilization as compared to when cells were permeabilized before fixation. Binding of both pol y clonal and monoclonal antibodies, as assessed by indirect immunofluo rescence microscopy in digitonin permeabilized cells, was inhibited by preincubation with rat liver cytosol. This inhibition was not observe d with antibody against another membrane protein (p28) with a cytoplas mically exposed epitope also residing in the Golgi/intermediate compar tment. Rabbit reticulocyte Lysate had a similar effect while Schizosac charomyces pombe cytosol inhibited binding to a greater degree than Sa ccharomyces cerevisiae cytosol. This inhibition by cytosol was prevent ed by coincubation with the antibody and was dose-dependent on the cyt osol. Inhibition did not occur on ice or at 15 degrees C, or when the cytosol was energy-depleted by apyrase treatment. Interestingly, pretr eatment of permeabilized cells with N-ethylmaleimide or its addition i nto the incubation mixture abolished inhibition. N-ethylmaleimide-trea ted cytosol, however, remained inhibitory. The findings suggest the ex istence of cytosolic factor (s) which interacts specifically with the cytoplasmic C-terminus of the KDEL receptor, which are likely to be co mponents of the KDEL protein retrieval machinery.