Incardona J P, Rosenberry T L
Department of Genetics, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, USA.
Mol Biol Cell. 1996 Apr;7(4):613-30. doi: 10.1091/mbc.7.4.613.
Drosophila has a single glycoinositol phospholipid (GPI)-anchored form of acetylcholinesterase (AChE) encoded by the Ace locus. To assess the role that GPI plays in the physiology, of AChE, we have replaced the wild-type GPI-AChE with a chimeric transmembrane form (TM-AChE) in the nervous system of the fly. Ace null alleles provided a genetic background completely lacking in endogenous GPI-AChE, and Ace minigene P transposon constructs were used to express both GPI- and TM-AChE forms in the tissues where AChE is normally expressed. Control experiments with the GPI-AChE minigene demonstrated a threshold between 9 and 12% of normal AChE activity for adult viability. Ace mutant flies were rescued by GPI-AChE minigene lines that expressed 12-40% of normal activity and were essentially unchanged from wild-type flies in behavior. TM-AChE minigene lines were able to rescue Ace null alleles, although with a slightly higher threshold than that for GPI-AChE. Although rescued flies expressing GPI-AChE at a level of 12% of normal activity were viable, flies expressing 13-16% of normal activity from the TM-AChE transgene died shortly after eclosion. Flies expressing TM-AChE at about 30% of normal levels were essentially unchanged from wild-type flies in gross behavior but had a reduced lifespan secondary to subtle coordination defects. These flies also showed reduced locomotor activity and performed poorly in a grooming assay. However, light level and electron microscopic immunocytochemistry showed no differences in the localization of GPI- and TM-AChE. Furthermore, endogenous and ectopic-induced expression of both AChEs in epithelial tissues of the adult and embryo, respectively, showed that they were sorted identically. Most epithelial cells sorted GPI- and TM-AChE to the apical surface, but cuticle-secreting epithelia sorted both proteins basolaterally. Our data suggest that rather than having a primary role in protein sorting, the GPI anchor or AChE plays some other more subtle cellular role in neuronal physiology.
果蝇有一种由Ace基因座编码的单一糖基磷脂酰肌醇（GPI）锚定形式的乙酰胆碱酯酶（AChE）。为了评估GPI在AChE生理学中的作用，我们在果蝇的神经系统中用嵌合跨膜形式（TM-AChE）取代了野生型GPI-AChE。Ace无效等位基因提供了一个完全缺乏内源性GPI-AChE的遗传背景，并且Ace小基因P转座子构建体用于在AChE正常表达的组织中表达GPI-和TM-AChE形式。用GPI-AChE小基因进行的对照实验表明，对于成年果蝇的存活能力，正常AChE活性的阈值在9%至12%之间。Ace突变果蝇可被表达正常活性12%至40%的GPI-AChE小基因系挽救，并且在行为上与野生型果蝇基本没有变化。TM-AChE小基因系能够挽救Ace无效等位基因，尽管其阈值略高于GPI-AChE。虽然表达正常活性12%的GPI-AChE的挽救果蝇是存活的，但表达来自TM-AChE转基因正常活性13%至16%的果蝇在羽化后不久死亡。以正常水平约30%表达TM-AChE的果蝇在总体行为上与野生型果蝇基本没有变化，但由于细微的协调缺陷而寿命缩短。这些果蝇还表现出运动活性降低，并且在梳理试验中表现不佳。然而，光镜和电镜免疫细胞化学显示GPI-和TM-AChE的定位没有差异。此外，分别在成年和胚胎的上皮组织中内源性和异位诱导表达的两种AChE表明它们的分选方式相同。大多数上皮细胞将GPI-和TM-AChE分选到顶端表面，但分泌角质层的上皮细胞将两种蛋白质分选到基底外侧。我们的数据表明，GPI锚或AChE在蛋白质分选中并非起主要作用，而是在神经元生理学中发挥一些其他更细微的细胞作用。
