photoreceptor cells.
Puneet Juneja1, Valerie Panneels2, Irmgard Sinning2, Wolfram Welte1
1 Department of Biology, University of Konstanz, D-78457 Konstanz, Germany
2 Heidelberg University Biochemistry Center, University of Heidelberg, Germany
4.1 Introduction
Nicotinic acetylcholine receptors (nAChRs) are crucial for signal transmission at inter-neuronal and inter-neuronal muscular junctions. Among them alpha 7 nAChRs play a vital role in calcium dependent signal transduction (94). They form a homopentamer of five identical subunits. The alpha 7 nAChRs are mainly localized in brain, spleen and lymphocytes. Like many other nAChR they open in response to acetylcholine but specifically form calcium channels. The alpha 7 nAChRs functions have been implicated in many diseases such as rheumatoid arthritis and Alzheimer’s disease (95–97). They are binder of snake toxins such as bungarotoxin and conotoxin like other nAChRs (98, 99). A structure of alpha 7 nAChR will help to understand the molecular mechanism and its function. Till now there is no stable expression system available for large-scale expression. We attempted to use the UAS-GAL4 expression system (5–7) targeting protein expression in drosophila eyes. Here we target the expression of the receptor in Drosophila photoreceptor cells (PRCs) and used the availability of large membrane invaginations (rhabdomeres), which provide a higher surface area for receptor integration in membrane. The GAL4 protein is a yeast transcription factor and is expressed under the control of drosophila specific promoters such as Glass multiple reporter (GMR) in the eyes of drosophila. The GAL4 protein binds on expression to the upstream activation sequence of the gene of interest to be expressed and regulates transcription (Figure 1).
Successful expression of membrane proteins with UAS-GAL4 system has been shown before by (100, 101).
4.2 Experimental procedures
Expression of Rat alpha 7 nAChR in Drosophila eyes
Rat alpha7 (Ra7) nAChR was cloned in the vector pUAST between EcoR1 and Xba1 sites with a N-terminal Flag-tag without the signal sequence. The complete UAS-Ra7 element was transposed from the vector pUAST in to the genomic DNA of Drosophila and was balanced in chromosome III by Tm3 Ser (serrate) marker. Our collaboration partners Dr Valerie Panneels and Prof Irmgard Sinning at the University of Heidelberg provided this expression system.
Stable expression system
To make a stable expression system, Drosophila with UAS-Ra7 element +/+; Ra7/ Tm3 was crossed with balancer flies Cyo/Br ; Tm2/Tm6 and flies with +/Cyo; Ra7/Tm3 phenotypes were selected. Similarly driver flies with an eye specific driver GMR-GAL4, GMR/GMR ; +/+ were crossed with balancer flies Cyo/Br ; Tm2/Tm6 and flies with GMR/Br ; +/Tm6 phenotype were selected. These selected phenotypes of driver flies and Ra7 flies of F1 generation were crossed together to get a stable drosophila F2 generation with GMR/Cyo ; Ra7/Tm6 as phenotypic marker.
Membrane preparation
Flies were reared at 18°C and 60% humidity on a standard fly food (yeast, corn syrup and agar) in 12 hours light/12 hours dark cycle. The flies were anaesthetized under CO2 and harvested in a falcon suspended in liquid nitrogen. Fly heads were separated from fly body using meshes of different size (Neo Labs Art. Nr 6-2382) and were suspended in membrane preparation buffer, 50 mM Tris, 250 mM sucrose, 120 mM KCl, 1mM EDTA, 5mM MgCl2, pH 8.2 in presence of protease inhibitors (Complete Roche) and 1mM PMSF. For 1 ml of heads 10 ml of membrane preparation buffer was used. Heads were homogenized in a glass teflon homogenizer with 20 strokes and centrifuged for 10 minutes at 1000g to remove unbroken cells and debris. The homogenization was repeated with the pellet in the same buffer and followed by centrifugation for 10 minutes at 1000 g. The supernatant was pooled and centrifuged for 1 hour at 120,000 g to collect the membrane pellet. Membranes were resuspended in the solubilization buffer 50 mM Tris, 250 mM KCl, 1mM EDTA, 5mM MgCl2, pH 8.2. Expression was checked with Western
blot with antibodies against Flag-tag (Figure2).
Solubilization and Affinity Purification
Membrane preparations at a protein concentration of 5 mg/ml were used for solubilization with different detergents Dodecyl maltoside (DDM) (2% w/v), Cymal-6 (2% w/v), Nonyl Maltoside (2% w/v), Zwittergent 3-14 (2% w/v), Zwittergent 3-10 (3%
w/v), Fos-choline-12 (2% w/v), CHAPS (2% w/v), LDAO (2% w/v). Membranes were incubated with the respective detergent concentrations at 7-10 °C in the cold room for 1 hour and later centrifuged at 120,000 g for 1 hour. The supernatant was used for Western blot with Anti Flag-tag antibodies to check solubilization of the nAChR (Figure 3). The membrane protein solubilized in Fos-choline-12 was passed over 0.5 ml Flag-tag affinity column, washed with 10 column volumes of solubilization buffer in 0.09% Fos-choline-12 and eluted with Flag peptide.
4.3 Results and Discussion
Heterologous expression of eukaryotic proteins is relatively difficult and impossible in E.coli due to absence of proper posttranslational machinery such as glycosylation and phosphorylation. Many eukaryotic membrane proteins do not fold and form inclusion bodies in E.coli. Here we use Drosophila as organism for heterologous expression of the rat alpha7 nAChR receptor. An eye specific driver Glass multiple reporter (GMR) was used for expression of rat alpha7 receptor in the Drosophila eyes. A Drosophila eye is composed of 750 cells called ommatidia, and each ommatidium consists of eight photoreceptor cells that have membrane proliferation (rhabdomere) which provide extensive surface for the insertion of membrane protein and have an appropiate machinery for the insertion of membrane proteins such as rhodopsin.
A stable expression line of transgenic flies expressing rat alpha7 was created and initial expression was checked with Anti Flag-tag antibody in whole head preparations and membranes preparations (Figure 2). A detergent screen showed that the membrane proteins were soluble in detergents Fos-choline-12, Zwittergent 3-10 and CHAPS, checked with Western blot against Flag-tag. The protein solublized in Fos-choline-12 was used for purification over Flag-tag affinity column. A small band appeared in the eluted fraction but it was not reproducible. Later investigation using control flies showed that
the Flag-tag antibodies were binding nonspecifically to a protein of apparent mass near 45 kDa (Figure 4). These bands were identified by mass spectroscopy to be an ATP Synthase beta subunit and an Actin protein (Figure 5). Presumably the expression of rat alpha7 nAChR was too low too be detected, or rat alpha 7 was not expressed at all; as a consequence the project was closed.
Figures
FIGURE 1. Representation of UAS-GAL4 expression system. Yeast transcriptional factor GAL4 is expressed under GMR in eyes (photoreceptor cells, PCRs). The GAL4 binds to UAS sequence of the gene to be expressed. The driver flies containing GMR-GAL4 element are crossed with flies that carry the gene of interest.
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FIGURE 2. Western blot with Anti-Flag tag antibodies for whole Drosophila heads and membrane preparations. Lane 1, Head preparation. Lane 2, membrane preparations and corresponding SDS PAGE (right)
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FIGURE 3. Purification of rat alpha7 nAChR on Flag affinity resin. Lane 1, Membrane preparation. Lane 2, solubilized fraction in Fos-choline-12. Lane 3 -5, flow-through and washing fraction. Lane 6, fractions eluted with the flag peptide.
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FIGURE 4. Expression in different control flies.
Western blot for head preparations of different flies, checked for expression of rat alpha 7 receptor using Anti-flag antibodies. Lane 1, GMR flies. Lane 2, Balancer flies. Lane 3, Ra 7 flies. Lane 4, Expression Flies. Along with expression flies, GMR and Balancer flies also show binding to Anti-flag antibodies.
FIGURE 5. SDS- PAGE of the membrane preparations from expression flies of Rat alpha 7 nAChR. Band 1 and Band 2 were analyzed by Mass Spectroscopy and found to be an ATP Synthase beta subunit and an Actin protein respectively.
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5. Mechanistic implications for the chorismatase FkbO based on the