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

Most members of the TransMEMbrane protein 16 (TMEM16) family are Ca2+-regulated scramblases that facilitate the bidirectional movement of phospholipids across membranes necessary for diverse physiological processes. The nhTMEM16 scramblase (from the fungus Nectria hematococca) is a homodimer with a...

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Bibliographic Details
Published in:The Journal of biological chemistry 2018-08, Vol.293 (31), p.12248-12258
Main Authors: Andra, Kiran K., Dorsey, Savanna, Royer, Catherine A., Menon, Anant K.
Format: Article
Language:English
Subjects:
ACP tag
Anoctamins - chemistry
Anoctamins - genetics
Anoctamins - metabolism
Biological Transport
calcium
Calcium - metabolism
Cell Membrane - chemistry
Cell Membrane - enzymology
Cell Membrane - metabolism
Dimerization
dithionite
Fluorescence
fluorescence lifetime imaging (FLIM)
fluorescence resonance energy transfer (FRET)
Fungal Proteins - chemistry
Fungal Proteins - genetics
Fungal Proteins - metabolism
glycerophospholipid
ion channel
Lipid Bilayers - chemistry
Lipid Bilayers - metabolism
liposome
MCP tag
Membrane Biology
membrane reconstitution
Nectria - chemistry
Nectria - enzymology
Nectria - genetics
Phospholipid Transfer Proteins - chemistry
Phospholipid Transfer Proteins - genetics
Phospholipid Transfer Proteins - metabolism
scramblase
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Summary:Most members of the TransMEMbrane protein 16 (TMEM16) family are Ca2+-regulated scramblases that facilitate the bidirectional movement of phospholipids across membranes necessary for diverse physiological processes. The nhTMEM16 scramblase (from the fungus Nectria hematococca) 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 Ca2+ 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 Ca2+-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 Ca2+-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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.RA118.003648