The amphipathic helix of an enzyme that regulates phosphatidylcholine synthesis remodels membranes into highly curved nanotubules

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Abstract

CTP:phosphocholine cytidylyltransferase (CCT) is an amphitropic protein regulating phosphatidylcholine synthesis. Lipid-induced folding of its amphipathic helical (AH) membrane-binding domain activates the enzyme. In this study we examined the membrane deforming property of CCT in vitro by monitoring conversion of vesicles to tubules, using transmission electron microscopy. Vesicle tubulation was proportional to the membrane density of CCT and proceeded either as growth from a pre-formed surface bud, or as a global transformation of roughly spherical vesicles into progressively thinner tubules. The tubulation pathway depended on the lipid compositional heterogeneity of the vesicles, with heterogeneous mixtures supporting the bud-extension pathway. Co-existence of vesicles alongside thick and thin tubules suggested that CCT can discriminate between flat membrane surfaces and those with emerging curvature, binding preferentially to the latter. Thin tubules had a limiting diameter of ~ 12 nm, likely representing bilayer cylinders with a very high density of 1 CCT/50 lipids. The AH segment was necessary and sufficient for tubulation. AH regions from diverse CCT sources, including C. elegans, had tubulation activity that correlated with α-helical length. The AH motifs in CCT and the Parkinson's-related protein, α-synuclein, have similar features, however the CCT AH was more effective in its membrane remodeling function. That CCT can deform vesicles of physiologically relevant composition suggests that CCT binding to membranes may initiate deformations required for organelle morphogenesis and at the same time stimulate synthesis of the PC required for the development of these regions.

Graphical abstract

Highlights

► Membrane binding activates CCT and also locally remodels the bilayer. ► The CCT amphipathic helix is necessary and sufficient for lipid vesicle tubulation. ► Nanotubes 11–12 nm in diameter can form with a density of 1 CCT/50 lipids. ► Pathways for nanotube generation are variable, and depend on vesicle lipid composition. ► CCT tubulation efficiency scales with amphipathic helix length, not binding affinity.

Abbreviations

CCT
CTP:phosphocholine cytidylyltransferase
AH
amphipathic helix
NLS
nuclear localization sequence
DMPC
1,2-dimyristoyl-sn-glycerophosphocholine
DMPC
1,2-dimyristoyl-sn-glycerophosphoglycerol (sodium salt)
DAG
diacylglycerol
POPS
1-palmytoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (sodium salt)
POPE
1-palmitoyl-2-oleoyl-sn-glycero-3-phospholethanolamine
PI
bovine liver phosphatidyl inositol
SM
egg sphingomyelin
DTT
1,4-dithio-d-threitol
SUVs
sonicated unilamellar vesicles
MLVs
multilamellar vesicles
LUVs
large unilamellar vesicles
SLVs
sucrose-loaded vesicles
NE
nuclear envelope
PM
phosphomimic
NE mix
LUVs composed of egg PC/POPE/PI /POPS/SM (55/20/10/10/5), PBS, phosphate buffered saline
TEM
transmission electron microscopy

Keywords

CTP:phosphocholine cytidylyltransferase
Membrane curvature
Vesicle tubulation
Electron microscopy

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