Structural mapping of fluorescently-tagged, functional nhTMEM16 scramblase in a lipid bilayer

Abstract

Most members of the TransMEMbrane protein 16 (TMEM16) family are Ca-regulated scramblases that facilitate the bidirectional movement of phospholipids across membranes necessary for diverse physiological processes. The nhTMEM16 scramblase (from the fungus ) is a homodimer with a large cytoplasmic region and a hydrophilic, membrane-exposed groove in each monomer. The groove provides the transbilayer conduit for lipids, but the mechanism by which Ca regulates it is not clear. Because fusion of large protein tags at either the N or C terminus abolishes nhTMEM16 activity, we hypothesized that its cytoplasmic portion containing both termini may regulate lipid translocation via a Ca-dependent conformational change. To test this hypothesis, here we used fluorescence methods to map key distances within the nhTMEM16 homodimer and between its termini and the membrane. To this end, we developed functional nhTMEM16 variants bearing an acyl carrier protein (ACP) tag at one or both of the termini. These constructs were fluorescently labeled by ACP synthase-mediated insertion of CoA-conjugated fluorophores and reconstituted into vesicles containing fluorescent lipids to obtain the distance of closest approach between the labeled tag and the membrane via FRET. Fluorescence lifetime measurements with phasor analysis were used to determine the distance between the N and C termini of partnering monomers in the nhTMEM16 homodimer. We now report that the measured distances do not vary significantly between Ca-replete and EGTA-treated samples, indicating that whereas the cytoplasmic portion of the protein is important for function, it does not appear to regulate scramblase activity via a detectable conformational change.

Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

“…1b), making them an obvious choice for fluorescence microscopy applications 30 . We recently showed that ATTO488 can be rendered non-fluorescent by chemical reduction with the membrane-impermeant dianion dithionite 31 , suggesting that ATTO488-modified phospholipids could be suitable reporters of scramblase activity in microscopy-based assays. We tested this possibility in a cuvette-based assay as follows: Proteoliposomes were reconstituted using egg phosphatidylcholine, trace amounts of ATTO488-PE and biotinylated phosphatidylethanolamine (biotin-PE), and Triton X-100-solubilized yeast ER membrane proteins ('Triton Extract' or TE), as described previously 9,11 .…”

Dysregulated calcium homeostasis prevents plasma membrane repair in Anoctamin 5/TMEM16E-deficient patient muscle cells

“…ANO5/TMEM16E/GDD1 (henceforth ANO5) belongs to the ten-member human Anoctamin/TMEM16 membrane protein family that includes Ca 2+ -activated ion channels (ANO1/TMEM16A, ANO2/TMEM16B) and phospholipid scramblases (ANO3, ANO4, ANO6, ANO7, and ANO10) 6,7 . In reconstitution-based assays ANO6/TMEM16F and its fungal homologues Aspergillus fumigatus TMEM16 (afTMEM16) and Nectria haematococca TMEM16 (nhTMEM16) show both scramblase and relatively unselective ion channel activity that are regulated by Ca 2+ 8–16 .…”

Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

“…1 b), making them an obvious choice for fluorescence microscopy applications 35 . We recently showed that ATTO488 can be rendered non-fluorescent by chemical reduction with the membrane-impermeant dianion dithionite 36 , suggesting that ATTO488-modified phospholipids could be suitable reporters of scramblase activity in microscopy-based assays. We tested this possibility in a cuvette-based assay as follows.…”