The collagen-tailed or asymmetric forms (A) represent a major component of acetylcholinesterase (AChE) in the neuromuscular junction of higher vertebrates. They are hetero-oligomeric molecules, in which tetramers of catalytic subunits of type T (AChE T ) are attached to the subunits of a triple-stranded collagen "tail." We report the cloning of a rat AChE-associated collagen subunit, Q. We show that collagen tails are encoded by a single gene, COLQ. The ColQ subunits form homotrimers and readily form collagen-tailed AChE, when coexpressed with rat AChE T . We found that the same ColQ subunits are incorporated, in vivo, in asymmetric forms of both AChE and butyrylcholinesterase. A splice variant from the COLQ gene encodes a proline-rich AChE attachment domain without the collagen domain but does not represent the membrane anchor of the brain tetramer. The COLQ gene is expressed in cholinergic tissues, brain, muscle, and heart, and also in noncholinergic tissues such as lung and testis.Acetylcholinesterase (AChE, EC 3.1.1.7)1 is highly concentrated at vertebrate neuromuscular junctions. This enzyme is encoded by a single gene, and adult mammalian muscles express a single splice variant, corresponding to the catalytic subunit of type T (AChE T ) (1, 2). At the post-translational level, however, quaternary interactions introduce a considerable diversity of molecular forms that are characterized by distinct localizations in cellular structures. These molecules include amphiphilic monomers (G 1 a ) and dimers (G 2 a ), nonamphiphilic tetramers (G 4 na ), as well as hetero-oligomeric structures in which tetramers of catalytic subunits are disulfide-linked with a hydrophobic "tail" (20 kDa) in the membrane-bound G 4 a forms (3, 4) or with a collagenous "tail" in the collagen-tailed or asymmetric (A) forms. The latter molecules consist of one, two, or three tetramers (A 4 , A 8 , A 12 ), which are disulfide-linked to the strands of the triple helical collagen tail (see Fig. 1A). G 1 a and G 2 a forms appear to remain mostly intracellular and represent precursors of more complex molecules. The G 4 na form is secreted and hydrophobic-tailed tetramers (G 4 a ) are attached to the plasma membrane. The collagen-tailed molecules are tethered in the basal lamina, and are largely responsible for the high concentration of AChE at the neuromuscular junction.To understand the biosynthesis of the various AChE forms and its regulation, it is necessary to analyze the association of AChE T catalytic subunits with anchoring subunits, particularly the collagen subunits, which have been named Q, according to the nomenclature of AChE-associated proteins (5). Cloning and expression of the collagen tail subunit of the asymmetric AChE forms from Torpedo electric organ (tQ 1 ) allowed us to show that the structural and catalytic subunits assemble into collagen-tailed molecules when coexpressed in COS cells (6). The primary sequence of the Q subunit comprises an N-terminal region, Q N , a collagen domain, and a C-terminal domain, Q C . We showe...