GTP-triggered fusion of rat liver microsomes derived from the endoplasmic reticulum was studied as a model for intracellular membrane fusion. We showed that the fusion is insensitive to pretreatment of microsomes with ATP and that the fusion of N-ethylmaleimide (NEM)-inactivated microsomes can not be restored by the addition of untreated cytosol. Treatment of microsomes with high salt did not inhibit fusion. These results are in contrast to those obtained in studies of reconstructed secretory and endocytic steps mediated by membrane fusion and suggest that the fusion of microsomes involves different proteins or combinations of proteins. Similar results have been obtained studying membrane fusion in the sea urchin egg cortical reaction. The NEM-sensitive fusion-related thiol groups in the membrane-bound microsomal fusion machinery have been characterized using bulky reagents prepared by the introduction of 3- (2-pyridyldithio)propionate (PDP) groups into BSA or aminodextrans. The reagents inhibited microsome fusion by reacting with thiol groups. The reaction was reversed by dithiothreitol. It has been found that modification of microsomes with these reagents protected fusion activity against inhibition by NEM. This indicates that fusion-related thiol groups are located on the outer side of the microsomal membrane and are accessible to macromolecules from the medium and, perhaps, to components of the opposite membrane in membrane fusion. The possibility that the exposed thiols belong to the tyrosine phosphatase on the microsome surface, known to have a highly exposed thiol group critical for its activity and suggested to have a regulatory role in assembly and disassembly of the endoplasmic reticulum, was examined. Fusion was completely inhibited by micromolar concentrations of zinc inactivating the phosphatase. However, it was insensitive to pre-treatment of microsomes with a recombinant soluble tyrosine phosphatase and to the presence of an excess of tyrosine phosphate in the medium. The application of the reagents to the sea urchin egg exocytotic apparatus gave very similar results. Based on these findings, we synthesized a reagent suitable for isolation of modified proteins. For this purpose, dinitrophenol (DNP) groups recognized by specific antibodies were introduced into BSA along with PDP groups. Immunoprecipitation of microsome proteins modified by BSA-PDP-DNP by anti-DNP immobilized monoclonal antibodies revealed a few proteins. Currently, raising monoclonal antibodies against immunopurified microsomal and sea urchin proteins reactive with BSA-PDP-DNP is in progress.
