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Two acetylcholinesterase (AcChoEase) polypeptide chains, alpha and beta, are expressed in avian nerves and muscles with apparent molecular masses of 110 and 100 kDa, respectively. We now show that individual quails express alpha, beta, or both AcChoEase polypeptide chains. By mating studies we show that the two AcChoEase polypeptides are autosomal and segregate as codominant alleles in classical Mendelian fashion. Biochemical studies of the two allelic AcChoEase polypeptides indicate that they have the same turnover number, have the same Km for acetylcholine, are immunoprecipitated to the same extent with a monoclonal anti-AcChoEase antibody, and can assemble with equal efficiency into multimeric forms. Thus there are no obvious functional differences between the two alleles. In heterozygotes, the rates of synthesis of the two polypeptides are identical, suggesting that there are no differences in expression of these two genes. Within an individual, nerves and muscles always express the same AcChoEase forms isolated from muscle indicates that all AcChoEase forms are comprised of the same allelic polypeptide chains. In contrast to the nicotinic acetylcholine receptors that appear to be encoded by complex multigene families, our studies on AcChoEase show that all forms of this important synaptic component in electrically excitable cells are encoded by a single gene. Thus differences in assembly and localization of the multiple synaptic forms of AcChoEase must arise through posttranscriptional events, posttranslational modifications of a similar AcChoEase polypeptide chain or both.

The major molecular form of acetylcholinesterase (AChE) from chicken brain is a membrane-bound glycoprotein with an apparent sedimentation coefficient of 11.4 S. Analysis of the purified protein by gel filtration, velocity sedimentation, and sodium dodecyl sulfate-gel electrophoresis shows that the solubilized enzyme is a globular tetramer with an apparent Mr = 420,000. This membrane-bound form of AChE is hydrophobic and readily aggregates in the absence of detergent. These aggregates are concentration-dependent, relatively stable in the presence of high salt concentrations, yet readily dissociate upon addition of detergent to the 11.4 S form, indicating that the interactions are hydrophobic. Polyclonal and monoclonal antibodies raised against chicken brain AChE purified by ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis precipitate AChE enzyme activity. However, these antibodies do not cross-react with the enzyme from chicken muscle which preferentially hydrolyses butyrylcholine. Immunoprecipitation of isotopically labeled enzyme molecules from tissue cultured brain cells and analysis by sodium dodecyl sulfate-gel electrophoresis shows that AChE consists of two polypeptide chains with apparent Mr = 105,000 (alpha) and 100,000 (beta) in a 1:1 ratio. Immunoblotting of brain AChE with either the polyclonal or monoclonal antibodies indicates that the alpha and beta chains share antigenic determinants. Furthermore, both polypeptide chains can be labeled with [3H]diisopropyl fluorophosphate, indicating that they each contain a catalytic site. This is the first indication that globular forms of AChE may consist of multiple polypeptide chains.

The synthesis, assembly, and processing of the multiple molecular forms of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) in quail muscle cultures was studied by using lectins to distinguish enzyme molecules residing in different subcellular compartments. Special emphasis was given to the assembly of asymmetric AcChoEase molecules because these appear to be the predominant, if not unique, forms of AcChoEase at the vertebrate neuromuscular junction. All cell surface and secreted AcChoEase forms bind to immobilized wheat germ agglutinin, ricin, and concanavalin A, indicating that they have complex oligosaccharides. After treatment of muscle cells with a membrane-permeable irreversible AcChoEase inhibitor, there is a rapid reappearance of the globular monomeric, dimeric, and tetrameric AcChoEase forms. However, the collagen-tailed asymmetric form does not appear until about 90 min after treatment. Analysis of the AcChoEase oligosaccharides with lectins indicates maturation to complex forms over a 90-min period. A large fraction of the intracellular globular AcChoEase molecules bind only to concanavalin A, indicating that they are assembled in the rough endoplasmic reticulum. In contrast, all intracellular asymmetric AcChoEase binds to wheat germ agglutinin, and a significant fraction binds to ricin, indicating that this unique AcChoEase form is assembled from subunits that have previously acquired complex sugars. I conclude that assembly of asymmetric AcChoEase, hence acquisition of information specifying basal lamina localization, occurs in the Golgi apparatus.

Tissue-cultured chicken embryo muscle cells synthesize several molecular forms of acetylcholinesterase (AChE) which differ in oligomeric structure and fate as membrane-bound or secreted molecules. Using irreversible inhibitors to inactivate AChE molecules we show that muscle cells rapidly synthesize and assemble catalytically active oligomers which transit an obligatory pathway through the Golgi apparatus. These oligomers acquire complex oligosaccharides and are ultimately localized on the cell surface or secreted into the medium. Immunoprecipitation of isotopically labeled AChE shows that the oligomers are assembled shortly after synthesis from two allelic polypeptide chains. About two-thirds of the newly synthesized molecules are assembled into dimers and tetramers, and once assembled these forms do not interconvert. Comparison of newly synthesized catalytically active AChE molecules with isotopically labeled ones indicates that a large fraction of the immature molecules are catalytically inactive. Pulse-chase studies measuring both catalytic activity and isotopic labeling indicate that only the catalytically active oligomers are further processed by the cell, whereas inactive molecules are rapidly degraded intracellularly by an as yet unknown mechanism. Approximately 70-80% of the newly synthesized AChE molecules are degraded in this manner and do not transit the Golgi apparatus. These studies indicate that muscle cells synthesize an excess of this important synaptic component over that which is necessary for maintaining normal levels of this protein. In addition, these studies indicate the existence of an intracellular route of protein degradation which may function as a post-translational regulatory step in the control of exportable proteins.

We have examined the possible role of clathrin-coated vesicles (CVs) in the genesis of the sarcoplasmic reticulum (SR) in developing chick skeletal myotubes. Calsequestrin (CSQ) a luminal Ca2+binding protein of the terminal SR cisternae, is contained within the vesicle lumen of skeletal muscle CVs in substantial amounts, approximately four molecules/CV. Employing 3-day cultures of chick skeletal myotubes we demonstrate that after a 30-min labeling with [35S]methionine and cysteine, radioactivity in CSQ remains high in the CVs 45 min later and then declines, while labeled CSQ in the SR continues to rise. No CSQ appears to be secreted. All of the CSQ in both the CVs and SR is sensitive to the activity of endoglycosidase H, and a significant fraction also binds to wheat germ agglutinin. Based on these results, we discuss the hypothesis that a selective CV-mediated pathway exists in developing skeletal muscle cells for the transport of CSQ from the early/intermediate Golgi apparatus to the SR.

The abundance and distribution of acetylcholinesterase (AChE) oligomeric forms expressed in skeletal muscle is strongly dependent upon the activity state of the cells. In this study, we examined several stages of AChE biogenesis to determine which ones were regulated by muscle activity. Inhibiting spontaneous contraction of tissue-cultured quail myotubes with tetrodotoxin (TTX) reduces AChE activity by approximately 30% of the levels found in actively contracting cells. This decrease is due primarily to the loss of 20 S asymmetric (collagen-tailed) AChE from TTX-treated cultures and is reflected in reduced pool sizes for both cell surface and intracellular AChE molecules. Using monoclonal anti-AChE antibodies to immunoprecipitate and quantify isotopically labeled enzyme molecules, we show that AChE down-regulation by TTX is not mediated through changes in the rates of synthesis or degradation of AChE polypeptide chains. Newly synthesized AChE polypeptides acquire enzymatic activity at the same rate in TTX-treated cultures as in actively contracting cells, however, a larger percentage of catalytically active dimers and tetramers are secreted from TTX-treated cultures compared with controls. These results suggest that TTX-induced down-regulation of asymmetric AChE occurs at the level of assembly of globular AChE molecules with collagen-like tail subunits in the Golgi apparatus, rather than through changes in the availability of catalytic subunits. Thus, post-translational mechanisms appear to play an important role in regulating the abundance and distribution of this important synaptic component in skeletal muscle.

Heparin is capable of solubilizing a subset of collagen-tailed (A12) acetylcholinesterase (AChE) molecules from skeletal basal lamina (Rossi, S. G., and Rotundo, R. L. (1993) J. Biol. Chem. 268, 19152-19159). In the present study, we used tissue-cultured quail myotubes to show that, like adult fibers, neither heparin- nor high salt-containing buffers detached AChE molecules from cell-surface clusters. Prelabeling clustered AChE molecules with anti-AchE monoclonal antibody 1A2 followed by incubation in heparin-containing medium showed that there was no reduction in the number or size of preexisting AChE clusters. In contrast, incubation of myotubes with culture medium containing heparin for up to 4 days reversibly blocked the accumulation of new cell-surface AChE molecules without affecting the rate of AChE synthesis or assembly. Newly synthesized A12 AChE becomes tightly attached to the extracellular matrix following externalization. However, in the presence of heparin, blocking the initial interactions between A12 AChE and the extracellular matrix results in release of AChE into the medium with a t1/2 of approximately 3 h. Together, these results suggest that once A12 AChE is localized on the cell surface, initially attached via electrostatic interactions, additional factors or events are responsible for its selective and more permanent retention on the basal lamina.

Immunocytochemical studies with a monoclonal antibody show that acetylcholinesterase (AcChoEase; EC 3.1.1.7) is distributed in clusters along the fibers of cultured sympathetic neurons but is essentially absent from cell bodies. Although tissue-cultured sympathetic neurons synthesize several oligomeric forms of AcChoEase, only the hydrophobic globular (G4) form of AcChoEase is present within these clusters. This G4 form is asymmetrically distributed within neurons and is transported preferentially into nerve fibers following its synthesis in the cell bodies. Thus G4 is found in clusters on neurons and is readily distinguishable from the hydrophilic forms on the surfaces of myotubes. The association of a specialized form of AcChoEase in densities on neurons in culture indicates that neurons and myotubes have distinct mechanisms for localizing AcChoEase molecules on their surfaces and suggests that these two types of electrically excitable cells have different requirements for organizing synaptic components on their plasma membranes.

Tissue-cultured muscle cells synthesize several oligomeric forms of acetylcholinesterase (AChE) destined for the cell surface or secretion. Previous studies on the biogenesis of AChE polypeptide chains have shown that only a small fraction become assembled into catalytically active oligomers which transit the Golgi apparatus and acquire endoglycosidase H (endo H) resistance. Most of the AChE polypeptides remain endo H-sensitive and are rapidly degraded intracellularly. We now show that all newly synthesized AChE polypeptides are transported from the rough endoplasmic reticulum to the Golgi apparatus where they acquire N-acetylglucosamine. However, approximately 80% of these AChE polypeptides remain endo H-sensitive and are degraded intracellularly with a half-life of about 1.5 h by a mechanism which is insensitive to lysosomotropic agents. These endo H-sensitive AChE molecules can be chased into clathrin-coated vesicles and/or the sarcoplasmic reticulum prior to degradation. Pulse-chase studies of isotopically labeled or catalytically active AChE molecules suggest that there are at least two discreet populations of clathrin-coated vesicles which leave the Golgi, one whose origin is cis/medial and one whose origin is trans. These studies indicate the existence of a post-rough endoplasmic reticulum, non-lysosomal degradative pathway for intra-luminal proteins and suggest that post-translational events at the levels of protein sorting and degradation may play a role in regulating the abundance of exportable proteins.