Two distinct proteins are associated with tetrameric acetylcholinesterase on the cell surface

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduce d the disulfide bonds in purified bovine brain AChE and sequenced tryptic f ragments from bands in the 80-kDa region. We obtained sequences belonging t o at least two distinct proteins: the P protein and another protein that wa s not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned c DNA encoding the second protein in a cDNA-library from bovine substantia ni gra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. Ho wever, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA elimina ted cell surface AChE and decreased the level of amphiphilic tetramer in ce ll extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that ancho rs AChE in membranes, together with the hydrophobic P protein.