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, CCTα and -β2, 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 CCTβ2 for anionic membranes was weaker than CCTα by more than an order of magnitude. Using chimeric CCTs, insertion/deletion mutants, and truncated CCTs, we show that the stronger affinity of CCTα 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 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 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 that modulate membrane binding strength. The NLS reinforces, whereas the phosphorylated tail antagonizes the attraction of domain M for anionic membranes.

FIGURE 1.

CCTα and -β₂ have highly similar C and M domains but divergent N and P domains and different membrane affinities. A, domain structure and sequence conservation are shown. Rat CCTα and -β₂ sequence alignment and percent similarity were calculated using ClustalW. Domain boundaries are approximate. Structured regions shown as rectangles or cylinders and disordered regions as dashed lines. B, prediction of disordered/ordered segments using the server RONN (79). Outputs from other prediction programs appear in supplemental Fig. S2. C, domain M comparison is shown. Amphipathic α-helical M domains of the two CCT isoforms (residues 242–293) are represented as 11/3 helical wheel diagrams (80). D, binding analysis of His-CCTα and His-CCTβ₂ to SLVs composed of PC/PG (3:2) at 20 °C is shown. Data were compiled from two independent experiments and were fit using GraphPad Prism 4 to the equation % Bound = 100K_p [L]/(1 + K_p[L]) (42), where [L] is the concentration of accessible lipid (½ of total lipid). Top and bottom values were constrained to 100 and 0%, respectively.

FIGURE 2.

Both CCTβ₂ and -α are dimers with intersubunit contacts involving region N. A, BS³ reactions are shown. Purified His-tagged CCTα or β₂ (0.4 μm) was reacted with BS³ in the presence or absence of 2 mm PG SUVs. Reactions were prequenched (−) or quenched after 20 min at 37 °C (+). B, copper phenanthroline reactions are shown. Purified His-tagged CCTβ₂ or CCTβ₂ C34S (0.34 μm) were either added to 0.4 mm CuSO₄ and 1.2 mm phenanthroline (+) or untreated (−). The samples were electrophoresed on 10% polyacrylamide gels and stained with silver.

FIGURE 3.

The NLS is responsible for the differential membrane binding affinity of CCT isoforms. Binding analysis to SLVs composed of PC/PG (3:2) at 20 °C. A, His-tagged CCT isoforms and region N-swapped chimeras are shown. B, untagged CCT isoforms and NLS insertion/deletion mutants are shown. C, in vitro dephosphorylated, untagged CCT isoforms, and NLS insertion/deletion mutants are shown. D, region P truncation mutants are shown. Data were compiled from at least two independent experiments and were fit using GraphPad Prism 4 to the equation given in the legend to Fig. 1D. Top and bottom values were constrained to 100 and 0%, respectively.

FIGURE 4.

CCTβ₂ has a weaker anionic lipid activation response than CCTα. The specific activities (nmol of CDP-choline/min/μg of CCT) of purified untagged CCT isoforms were measured in parallel as a function of increasing concentrations of the indicated LUVs. The data were normalized to the maximal activities obtained in the presence of 3:2 PC/PG LUVs, (CCTα, 14.6 ± 1.1 nmol/min/μg; CCTβ₂, 10.1 ± 1.4 nmol/min/μg). Data represent the mean ± S.D. of four independent determinations. ■, CCTα; ▴, CCTβ₂.

FIGURE 5.

CCTβ₂ cannot tether lipid vesicles because it lacks a NLS. The increase in apparent absorbance (400 nm) of PG SUVs due to the addition of CCT was monitored for 3 min. The apparent absorbance due to vesicles alone was subtracted from the plateau value of each CCT concentration. The data represent the means ± S.E. or range for at least two independent determinations. A, His-tagged CCT isoforms and region N-swapped chimeras are shown. B, untagged CCTβ₂ and CCTβ+NLS are shown. Untagged CCTα was included as a positive control.

FIGURE 6.

Differential membrane partitioning of CCT isoforms to oleic acid-enriched cell membranes. COS-1 cells expressing CCT constructs at approximately equivalent levels were treated with OA/BSA in molar ratios of 3.3/1 to 266/1 for 1 h at 37 °C. Cells were harvested and fractionated, and the CCT activity units in each fraction were determined. The proportion of CCT in the membrane fraction versus the total CCT in the cytosol, membrane, and particulate fraction for each OA/BSA ratio was plotted. The individual data points from at least two independent determinations were fit to curves by GraphPad Prism 4 or fitted manually in the case of CCTβ₂. A, untagged full-length CCT isoforms and NLS insertion/deletion mutants are shown. B, region P truncated CCT isoforms and NLS insertion/deletion mutants are shown. C, equivalent proportions of the soluble (sol) and membrane (M) fractions of cells transfected with the indicated full-length CCTs were analyzed by immunoblot with an antibody against the conserved catalytic domain. Cells were untreated or treated with OA/BSA at a molar ratio of 66; this concentration is indicated in panel A with an arrow.