The Intrinsically Disordered Nuclear Localization Signal and Phosphorylation Segments Distinguish the Membrane Affinity of Two Cytidylyltransferase Isoforms

Abstract

Membrane phosphatidylcholine homeostasis is maintained in part by a sensing device in the key regulatory enzyme, CTP: phosphocholine cytidylyltransferase (CCT). CCT responds to decreases in membrane phosphatidylcholine content by reversible membrane binding and activation. Two prominent isoforms, CCT␣ and -␤ 2 , have nearly identical catalytic domains and very similar membrane binding amphipathic helical (M) domains but have divergent and structurally disordered N-terminal (N) and C-terminal phosphorylation (P) regions. We found that the binding affinity of purified CCT␤ 2 for anionic membranes was weaker than CCT␣ by more than an order of magnitude. Using chimeric CCTs, insertion/deletion mutants, and truncated CCTs, we show that the stronger affinity of CCT␣ can be attributed in large part to the electrostatic membrane binding function of the polybasic nuclear localization signal (NLS) motif, present in the unstructured N-terminal segment of CCT␣ but lacking in CCT␤ 2 . The membrane partitioning of CCT␤ 2 in cells enriched with the lipid activator, oleic acid, was also weaker than that of CCT␣ and was elevated by incorporation of the NLS motif. Thus, the polybasic NLS can function as a secondary membrane binding motif not only in vitro but in the context of cell membranes. A comparison of phosphorylated, dephosphorylated, and region P-truncated forms showed that the in vitro membrane affinity of CCT␤ 2 is more sensitive than CCT␣ to phosphorylation status, which antagonizes membrane binding of both isoforms. These data provide a model wherein the primary membrane binding motif, an amphipathic helical domain, works in collaboration with other intrinsically disordered segments that modulate membrane binding strength. The NLS reinforces, whereas the phosphorylated tail antagonizes the attraction of domain M for anionic membranes.

CTP:phosphocholine cytidylyltransferase: Function, regulation, and structure of an amphitropic enzyme required for membrane biogenesis

“…2). This basic structure is supported by bioinformatics analysis [69,71], and was confirmed by limited proteolysis [72,73], site-directed fluorescence anisotropy, and deuterium exchange/ mass spectrometry [74]. The regulatory tail domain is comprised of domain M, a membrane-induced amphipathic helix (m-AH) in the CCT mem form, followed by a disordered region P that contains up to 16 or 21 phosphorylation sites in mammalian CCTa [72,75,76] or CCT b 2 [69], respectively.…”

“…Rat CCTa purified from insect or mammalian cells is heterogeneously phosphorylated on all 16 serines in region P, with an average of 6-7 phosphoserines per polypeptide [72,75,76]. Purified rat CCTb2 also averages $7 phosphates per polypeptide [69]. Deletion of region P or the phosphorylation sites in P eliminated CCTa phosphorylation in cells, indicating that phosphorylation is restricted to this portion [75,168,169].…”

“…A homodimeric structure, demonstrated for rat CCTa and b2 [68,69], is the norm for members of the cytidylyltransferase family [23,70]. Each monomer has a globular head region housing the catalytic domain and a structurally loose regulatory tail domain (Fig.…”

Structural Basis for Autoinhibition of CTP:Phosphocholine Cytidylyltransferase (CCT), the Regulatory Enzyme in Phosphatidylcholine Synthesis, by Its Membrane-binding Amphipathic Helix

“…Protease accessibility, photocross-linking, and site-directed fluorescence anisotropy suggested a bipartite structure for domain M in the CCT soluble form: an N-terminal disordered segment (residues ϳ235-270) followed by a more structured segment (residues 272-301) (20,21). This bipartite structure is also predicted by many disorder prediction algorithms and is a feature of CCT M domains across phyla (16,22).…”

“…We prepared pET14b CCT-312 as follows. Using pAX142-HisCCT␣-312 (22) as the template, the opening reading frame encoding CCT(1-312) was PCR-amplified with 5Ј-ATATCATATGGATGCACAGAGTT-CAG-3Ј and 5Ј-ATATGGATCCTCACTGCAGCATCCGA-3Ј oligonucleotide primers containing NdeI and BamHI restriction sites, respectively (sites in italics). The PCR fragment was double digested using NdeI/BamHI and cloned into similarly digested pET14b plasmid.…”