Long-term objectives are to characterize the biochemical features of distinct acetyl-cholinesterase (AChE) forms in synapses: their structures, processes of assembly and localization, and functions in synaptic transmission. The current program focuses on the structures that attach AChEs to cell membranes. Previous work has demonstrated three distinct classes of attachment structures, each rather unusual for membrane proteins; 1) Dodecameric (A12) AChE is localized in skeletal neuromuscular junctions by collagen-like subunits. 2) Dimeric (G2) AChE in mammalian erythrocytes, nerve endings in torpedo electric organ, and insect heads in anchored by a glycoinositol phospholipid covalently linked to the C-terminus. 3) Tetrameric (G4) AChE in mammalian brain appears to bind to membranes through a small hydrophobic noncatalytic subunit. The proposed Specific Aims involve the latter two AChE classes. 1. A 20-kDa subunit in G4 bovine brain AChE has been identified that appears responsible for membrane interaction, and its structure will be determined to establish whether it is a peptide a lipid, or both. The subunit amino acid sequence will be pursued, and antisera against this subunit will be obtained 2. Additional structural features of the glycoinositol phospholipid anchor of human erythrocyte AChE will be defined. These include the hexose and hexose phosphate components and the positions of hexose and inositol linkage. Peptides that include the C- terminus of this AChE will sequenced. 3. Palmitoylation of inositol renders the anchor of human erythrocyte AChE resistant to a characteristic phospholipase C. Evidence of this modification will be sought in a variety of cell lines by examining both endogenous G2 AChE and transfected Drosophila G2 AChE. Free glycoinositol phospholipid precursors of AChE anchors will be investigated. 4. A nucleotide sequence corresponding to a stable transmembrane peptide will be substituted for the 3' coding region that directs the glycoinositol phospholipid anchor addition in Drosophila AChE. Our long-term goal in this aim is to produce a transgenic fly in which AChE is anchored only by peptide and assess the effects of this changes on fly development and AChE location. Because 30- 40 membrane proteins currently are known to be anchored by glycoinositol phospholipids, further information about these anchors is vital to a better understanding of membrane protein function.
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