PMC

Disorder prediction of complete CCT protein sequences using DISOClust (). The diagram at the top shows the domain structure of a CCT monomer with rat CCTα numbering. All CCTs have disordered N and C termini, highly ordered catalytic (C) domains, and biphasic membrane-binding (M) domains. The gray-shaded box in each panel corresponds to the M domain. The sequence identifiers are provided in the legend to . N, N-terminal region; P, phosphorylation region.

Deletion of 22-mer segment decreases the response to activating lipids. Activities were measured in the presence of the indicated concentrations of egg PC/egg PG (1:1) sonicated vesicles (A), egg PC/egg PG (4:1) sonicated vesicles (B), or DOPC/DOPE (2:3) 100 nm extruded vesicles (C). Data are means ± S.E. of two independent determinations. △, WT rat CCTα; □, rat CCT-Δ22; ×, rat CCTα-236; SUV, small unilamellar vesicle; LUV, large unilamellar vesicle.

Probing interactions between catalytic head and regulatory tail in trans. A, rat CCTα catalytic domain does not capture its tail domain in trans. We measured the capture of a domain M peptide (top panel), M + P tail fragment (middle), or M + P phosphomimic (PM) fragment (bottom) onto nickel-agarose beads without (−) or with (+) His-tagged catalytic fragment (His-236). Samples were subjected to SDS-PAGE using 12% Tricine gels. The pellet fractions containing the nickel beads are shown. The numbers represent the density of the tail bands determined by ImageQuant. B, rat CCTα catalytic domain is not inhibited by its tail domain in trans. Catalytic fragment (CCT-236) was mixed with the indicated molar excess of tail fragment for 5 min prior to activity analysis. M + P, open bar; M + P(PM), filled bar. Data are the average of two independent determinations and are normalized to the activity obtained without tail fragment.

Lipid-dependent and -independent activity of CCT chimeras. CCT was activated by egg PC/oleic acid (1:1)-sonicated vesicles (A) or DOPC/DOPE (2:3) 100 nm extruded vesicles (B). R-worm refers to the rat catalytic domain fused to the regulatory tail of the CCT from C. elegans, etc. Data are means ± S.E. of four independent determinations; in many cases, the error bar is within the symbol. Symbols in B are defined in A. C, purity of CCT chimeras. ∼2 μg of protein purified by nickel-agarose chromatography was separated on 10% SDS-PAGE and stained with Coomassie Brilliant Blue-R. Δ22 refers to rat CCTΔ272–293. D, activity of all CCT constructs in the absence of lipid. Data are means ± average deviations of 4–7 independent determinations. *, p = 0.037. Inset, micrograms of pure CCT in the assay are plotted versus activity units (nanomoles of CDP-choline formed/min) for the yeast, synuclein, and Drosophila chimeras as well as the rat control CCT. Symbols are as in A. SUV, small unilamellar vesicle; LUV, large unilamellar vesicle.

Deletion of the 22-mer reduces CCT cell membrane affinity and accelerates PC synthesis. A, membrane partitioning in cells. COS cells expressing rat CCTα-WT, CCT-236, or CCT-Δ22 were fractionated, and the total activity units in each fraction were determined. The graph shows the ratio of units in the membrane fraction versus soluble fraction. The data are means ± range of two independent experiments. B, PC synthesis was monitored by a pulse-chase protocol for cells transfected with CCT: △, WT rat CCTα; □, rat CCT-Δ22; ×, rat CCTα-236. Data are means ± range of two independent determinations. The disintegration/min values in the lipid fraction from cells transfected with empty vector were determined in parallel, and these values were subtracted as base line. The disintegration/min values were normalized to the CCT expression levels, determined via immunoblot of equivalent cell lysate volumes using an antibody against the catalytic domain (inset). Lysates analyzed were from cells transfected with empty vector (P) or the indicated CCTs. Pure CCT standards are in the right lanes. The numbers on the blot are relative signal volumes. The upper band in each lane represents the CCT dimer, and the lower band represents the CCT monomer. Western analysis was repeated using different volumes of lysate, yielding similar results.

A, sequence alignment of the M region of CCT. The boundaries of M were chosen based on empirical evidence for insertion of Phe-234 of rat CCTα into lipid vesicles () and phosphorylation of rat CCTα beginning at residues Ser-315 (, ). Rat, α isoform, P19836; Dros (D. melanogaster) CCT-2, B4QMG1; C. elegans, Q3HKC4; yeast (S. cerevisiae), P13259. The alignment was done manually. There is discrepancy in translation products for the C. elegans CCT in the databanks due to three potential start sites. We use the start site associated with the clone characterized by Friesen et al. (). The blue-shaded box highlights the positively charged N-terminal segment, and the yellow-shaded box highlights the regulatory 22-mer segment containing 10–11 conserved hydrophobic amino acids. The four 11-mer repeats in rat CCTα are underlined. Previous literature refers to three 11-mer repeats in rat CCTs, based on a frame starting with ²⁵⁶VEEKS … (, , , , ). Amino acids are color-coded as follows: red, acidic and phosphoserine; blue, basic; green, hydrophilic; black, hydrophobic; orange, glycine, proline. B, physicochemical features of subsections of domain M. The sequences were analyzed with HeliQuest (), where the amino acid (aa) is the sequence range for the analysis; 〈H〉 is the mean hydrophobicity, and 〈μH〉 is the mean helical hydrophobic moment calculated using the Eisenberg formula (), based on the hydrophobicity scale of Fauchere and Pliska (). The output of the analysis as 3–11 helices is shown; analysis as canonical α-helices yielded only slight variation in 〈μH〉 values. C, sequence alignment of rat CCTα (P19836) with human α-synuclein (α-S; P37840.1). The 11-mer repeats are underlined, and the color coding is as for A.