The intrinsically disordered nuclear localization signal and phosphorylation segments distinguish the membrane binding affinity of two cytidylyltransferase isoforms

Membrane phosphatidylcholine (PC) homeostasis is maintained in part by a sensing device in the key regulatory enzyme, CTP: phosphocholine cytidylyltransferase (CCT). CCT responds to decreases in membrane PC content by reversible membrane binding and activation. Two prominent isoforms, CCTα and β2, have nearly identical catalytic domains and very similar membrane binding amphipathic helical (M) domains, but have divergent and structurally disordered amino-terminal (N) and carboxy-terminal phosphorylation (P) regions. I found that the anionic membrane binding affinity of purified CCTβ2 was weaker than CCTα by at least an order of magnitude. Using chimeric CCTs, insertion/deletion mutants and truncated CCTs I showed that the stronger affinity of CCTα can be attributed in large part to the secondary electrostatic membrane binding function of the polybasic nuclear localization signal (NLS) motif, present in the unstructured region of region N of CCTα, but lacking in CCTβ2. The membrane partitioning of CCTβ2 in cells enriched with the lipid activator, oleic acid, was also weaker than that of CCTα, 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 also in the context of cell membranes. A comparison of phosphorylated, dephosphorylated, and region P truncated forms showed that the in vitro membrane affinity of CCTβ2 is more sensitive than CCTα 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, which modulate membrane binding strength. The NLS reinforces, while the phosphorylated tail antagonizes the attraction of domain M for anionic membranes.