Abstract
The direct regulation of a mycobacterial adenylate cyclase (Rv1625c) via exchange of its membrane anchor by the quorum sensing receptor CqsS (Vibrio harveyi) has recently been reported (Beltz et al., 2016). This protocol describes the expression and membrane preparation for these chimeric proteins.
Keywords: Adenylate cyclase (AC), Quorum sensing (QS), CqsS, Membrane protein, Protein expression, French press
Background
Membrane-delimited mammalian adenylate cyclases (ACs) are class IIIa ACs. Regulation is indirectly via stimulatory (or inhibitory) Gα-proteins which are released intracellularly upon extracellular stimulation of G-protein-coupled-receptors (GPCR) by first messengers. ACs generate the universal second messenger cAMP using ATP as a substrate. The size of the two hexahelical membrane domains in vertebrate ACs by far exceeds the requirements for a simple membrane anchorage. Yet, regulatory features of this intrinsic membrane anchor/receptor domain are unknown. To investigate a potential function the canonical class IIIa AC Rv1625c from Mycobacteria was chosen which can be easily expressed in bacteria (Guo et al., 2001 and 2005) in contrast to mammalian AC isoforms. We replaced the hexahelical membrane anchor of the Rv1625c AC by the receptor domain of the hexahelical quorum sensing (QS) receptor from V. harveyi, CqsS, to examine whether we can confer a direct regulation of the AC by the QS-ligand ‘cholera autoinducer-1’, CAI-1. The design of the QS-receptor and the class IIIa membrane anchors are highly similar, i.e., minimal transmembrane α-helices and exceptionally short connecting loops.We have demonstrated a direct regulation of a class IIIa AC by an extracellular signal. This considerably supports the hypothesis of a receptor function for the membrane anchor. Indeed, it raises the possibility that in addition to the well-established indirect GPCR-Gα-protein regulation of mammalian ACs a second, rather different set of signals directly impinge on this most important enzyme.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
The expression of the proteins is verified by SDS-PAGE (Laemmli, 1970) and Western blot. The isolated membranes are incubated in 4x SDS-loading dye at room temperature for at least 30 min prior to application to SDS-PAGE (do not boil sample). Dilute the sample (membrane preparation) in MilliQ H2O to get 2.5-5 µg of protein in a final volume of 15 µl. Add 5 µl 4x SDS-loading dye. Load 20 µl of the mixture into one slot of the SDS-PAGE. The membranes are incubated for 1 h with each antibody (first antibody at 4 °C, second antibody at room temperature). The first antibody is either the RGSHis- (Figures 3A and 3C) or the S-tag-antibody (Figure 3B). In both cases, the ECL Plex goat-anti-mouse IgG-Cy3-antibody is used as a secondary antibody (dilution 1:2,500). Western blot evaluation is carried out using an Ettan DIGE Imager. Note: In contrast to soluble proteins, membrane proteins are NOT boiled (95 °C, 5-10 min). Figure 3. Western blots (A-C) and SDS-PAGE (D)
Recipes
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 766; TP B08).
References
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