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Genetic analysis of glycolipid anchor function using Drosophila acetylcholinesterase as a model protein
| Title: | Genetic analysis of glycolipid anchor function using Drosophila acetylcholinesterase as a model protein |
| Author: | Incardona, John Patrick |
| Description: | Glycoinositol phospholipid (GPI)-anchored proteins in many mammalian polarized epithelial cell lines are found on the apical surface, incorporated into cold Triton-insoluble complexes at the level of the trans-Golgi, and frequently found sequestered in cholesterol-dependent plasma membrane invaginations called caveolae. To assess the role of GPI anchoring in vivo in Drosophila, we have focused on acetylcholinesterase (AChE) encoded by the Ace locus on the third chromosome. We constructed secreted (SEC) and chimeric transmembrane (TM) forms of AChE and substituted these for the wild type CPI-anchored form in transgenic flies. Expression in a Drosophila cultured cell line (S2) showed that the biochemical properties of the mutants were not altered except as predicted. Control experiments with a variety of GPI-AChE transgene inserts expressing a range of AChE activities demonstrated a threshold of only about 10% of normal activity for adult viability; AChE levels of <5% of normal resulted in late embryonic lethality. ce mutant flies were rescued by GPI-AChE transgenes that expressed 12-40% of normal activity and were essentially unchanged from wild type flies in behavior. In contrast, no SEC1 transgene insert could rescue Ace null alleles, indicating that AChE must be me mbrane-bound to function appropriately in the fly nervous system. Our data suggested that secreted AChE is either degraded within neurons or is unstable in the extracellular space of neuropil. TM-AChE transgenes were able to rescue Ace null alleles, although with a slightly higher threshold than that for GPI-AChE. Animals expressing 15% of normal activity from a TM-AChE transgene showed an abnormal phenotype not observed in rescued flies expressing 12% of normal from a GPI-AChE transgene: most animals died shortly after eclosion and many were unable even to eclose. However, flies expressing TM-AChE at about 30% of normal levels were essentially unchanged from wild type flies in behavior and fertility but had a reduced lifespan secondary to subtle coordination defects and tended to be entrapped in the food. Light level and electron microscopic immunocytochemistry showed no differences in the localization of GPI- and TM-AChE. Furthermore, expression of both AChEs in epithelial tissues of the adult and embryo, respectively, showed that they were sorted identically, although the various tissues differed in their sorting behavior. Immunocytochemistry on primary cultures of CNS neurons with antibodies to GPI and TM AChE, GPI-anchored fasciclin I, and insect glyscosphingolipids showed discrete clusters of all antigens at both light and electron microscopic levels. Overlap of the clusters appeared to be random, however, and the clusters were not sensitive to sterol-binding drugs. Our data suggest that rather than having a primary role in protein sorting, the GPI anchor of AChE plays some other more subtle cellular role |
| Permanent Link: |
http://rave.ohiolink.edu/etdc/view?acc_num=case1061923759
http://hdl.handle.net/2374.OX/17508 |
| Date: | 1995 |
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