CELLULAR-DISTRIBUTION OF A GPI-ANCHORED COMPLEMENT REGULATORY PROTEINCD59 - HOMODIMERIZATION ON THE SURFACE OF HELA AND CDB9-TRANSFECTED CHO CELLS

Human glycosyl phosphatidylinositol-anchored protein CD59 was solubili zed in detergent-insoluble complexes (DICs) and in post-nuclear pellet s by a two-step solubilization procedure using Triton X-100 and octylg lucoside, CD59 molecules are recovered in both fractions, the amount b eing greater in the latter fraction in all cell types tested, Specific labeling of surface CD59 molecules revealed that the CD59 detected in DICs originated from intracellular compartments, whereas that in post -nuclear pellets was in part derived from the cell surface, Cross-link ing of surface proteins with chemical cross-linker followed by Western blotting with anti-CD59 antibody revealed cross-linked products with molecular masses of 28-36 kDa on HeLa and human CD59 cDNA-transfected CHO cells; the CD59-associating molecules were estimated to be 13-18 k Da in size, The cross-linked products were extracted in the post nucle ar pellets, and CD59 existed mainly as a cross-linked form on the cell surface, Two-dimensional electrophoresis of the cross-linked products revealed no trace of molecules other than CD59, The cross-linked prod ucts showed the same N-terminal sequences as CD59 and a strikingly sim ilar amino acid composition to that of CD59, Thus, most likely, the cr oss-linked products are CD59 dimers, The finding that CD59 localized o n outer membranes is all in the form of dimers suggests the importance of dimerization for CD59 functioning.