Study of subcellular localization of membrane‐bound choline acetyltransferase in Drosophila central nervous system and its association with membranes

Abstract

Choline acetyltransferase (ChAT), the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine, exists in a soluble and membrane‐bound form in cholinergic nerve terminals of different animal species. This study was performed on the enzyme present in Drosophila central nervous system. We show that the two forms of the enzyme have the same apparent molecular weight (75 kDa) when analysed by immunoblotting using an antibody we raised against the recombinant enzyme. According to different authors, membrane‐bound enzyme might be associated with synaptic vesicles or plasma membrane. Subfractionation of Drosophila head homogenates in linear glycerol gradients showed that ChAT does not associate with synaptic vesicles. Analysis of ChAT activity and immunoreactivity showed that two peaks of ChAT were produced. One peak was present in fractions containing soluble components and the other was associated with rapidly sedimenting membranes containing plasma membranes. ChAT in the first peak was mainly hydrophilic. A large proportion of ChAT associated with rapidly sedimenting membranes was amphiphilic. Further fractionation of these membranes by flotation in sucrose gradients showed that membrane‐associated ChAT sedimented in fractions containing plasma membrane marker. Membrane‐bound ChAT was neither solubilized nor converted to hydrophilic enzyme after membrane treatment with 1 m hydroxylamine, suggesting that the enzyme is not palmitoylated and therefore not anchored to membrane through thioester‐linked long chain fatty acid. Partial solubilization of ChAT present on membranes with urea and carbonate suggests that this form of ChAT is a peripheral membrane protein. Carbonate solubilization of membrane‐bound ChAT converted the enzyme from hydrophobic to hydrophilic protein.

Identification of Cholinergic Synaptic Transmission in the Insect Nervous System

“…[38][39][40][41][42][43][44] In Drosophila melanogaster, ChAT exists in cholinergic nerve terminals in at least two forms; a soluble and membrane-bound form that exhibit the same apparent molecular weight (75 kDa). 35 It should be noted that purification of locust ChAT results in a band at 65 kDa 45 very similar to that of reported in the spider Cupiennius salei. 46 The main reason that explains such difference may be that different forms of the enzyme exist.…”

“…Cholinergic neurons can be detected by (1) using antibodies against ChAT, (2) in situ hybridization to detect the transcript and (3) staining for a reporter gene fused to the regulatory sequence of the ChAT gene. [35][36][37] In this case, ChAT has been first identified in the larval neuropil, including the larval antennal and optic lobes and in the gustatory target regions, which appeared to be due to the terminals of cholinergic afferents. [38][39][40][41][42][43][44] In Drosophila melanogaster, ChAT exists in cholinergic nerve terminals in at least two forms; a soluble and membrane-bound form that exhibit the same apparent molecular weight (75 kDa).…”

Nuclear Localization of the 82-kDa Form of Human Choline Acetyltransferase

“…A major difference between Drosophila and human, however, is that the M transcript that encodes the 82-kDa form of human ChAT also encodes 69-kDa ChAT from a second translation initiation site. It appears, from the data that exist, that Drosophila ChAT is produced as a single 75-kDa protein (32). To date, there is no evidence to indicate that 82-kDa human ChAT is proteolytically cleaved to generate 69-kDa ChAT.…”

Functional Characterization of Phosphorylation of 69-kDa Human Choline Acetyltransferase at Serine 440 by Protein Kinase C

“…The means by which ChAT binds nonionically to membranes has not been elucidated; it does not have obvious hydrophobic domains for association with the lipid bilayer, and the presence of covalent modifications such as a glycosylphosphatidylinositol linkage have been controversial (16 -18). Eder-Colli and colleagues (19) reported recently that in Drosophila neurons amphiphilic ChAT had properties of a peripheral membrane protein and was removed from plasma membranes by alkaline carbonate (1 g) of purified wild-type or S440A-ChAT were phosphorylated in vitro using PKC, as described under "Experimental Procedures." At varying times up to 1 h, samples were removed from incubation and prepared for analysis by SDS-PAGE.…”