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• W2021513697 abstract "CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the N-terminal domain (amino acids 1–72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73–236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface. CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the N-terminal domain (amino acids 1–72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73–236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface. CTP:phosphocholine cytidylyltransferase (CCT) 1The abbreviations used are: CCT, CTP:phosphocholine cytidylyltransferase; AD, activation domain; BS3, bis(sulfosuccinimidyl)suberate; Cu(Phe)3, copper phenanthrolene; DBD, DNA binding domain; DSP, dithiobis(succinimidyl propionate); GCT, glycerolphosphate cytidylyltransferase; NEM, N-ethylmaleimide; PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PMSF, phenylmethylsulfonyl fluoride; WT, wild-type. 1The abbreviations used are: CCT, CTP:phosphocholine cytidylyltransferase; AD, activation domain; BS3, bis(sulfosuccinimidyl)suberate; Cu(Phe)3, copper phenanthrolene; DBD, DNA binding domain; DSP, dithiobis(succinimidyl propionate); GCT, glycerolphosphate cytidylyltransferase; NEM, N-ethylmaleimide; PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PMSF, phenylmethylsulfonyl fluoride; WT, wild-type. is a key regulatory enzyme in phosphatidylcholine (PC) biosynthesis. It is activated by reversible binding to cell membrane lipids (1Cornell R.B. Northwood I.C. Trends Biochem. Sci. 2000; 25: 441-447Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). The most potent lipid activators are anionic phospholipids and fatty acids (2Cornell R.B. Biochemistry. 1991; 30: 5873-5880Crossref PubMed Scopus (60) Google Scholar, 3Arnold R.S. Cornell R.B. Biochemistry. 1996; 35: 9917-9924Crossref PubMed Scopus (84) Google Scholar). Type II lipids such as diacylglycerol, unsaturated phosphatidylethanolamine, and oxidized PCs will also promote the binding and activation of this enzyme (3Arnold R.S. Cornell R.B. Biochemistry. 1996; 35: 9917-9924Crossref PubMed Scopus (84) Google Scholar, 4Davies S.M. Epand R.M. Kraayenhof R. Cornell R.B. Biochemistry. 2001; 40: 10522-10531Crossref PubMed Scopus (110) Google Scholar, 5Drobnies A.E. van Der Ende B. Thewalt J.L. Cornell R.B. Biochemistry. 1999; 38: 15606-15614Crossref PubMed Scopus (19) Google Scholar). It has been suggested that this regulatory mechanism enables CCT to respond to decreases in the relative PC content of cell membranes and thus maintain PC homeostasis (6Sleight R. Kent C. J. Biol. Chem. 1983; 258: 836-839Abstract Full Text PDF PubMed Google Scholar, 7Jamil H. Yao Z. Vance D.E. J. Biol. Chem. 1990; 265: 4332-4339Abstract Full Text PDF PubMed Google Scholar). The membrane binding domain has been mapped to an internal 50–60- residue amphipathic α-helix (8Craig L. Johnson J.E. Cornell R.B. J. Biol. Chem. 1994; 269: 3311-3317Abstract Full Text PDF PubMed Google Scholar, 9Dunne S.J. Cornell R.B. Johnson J.E. Glover N.R. Tracey A.S. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (89) Google Scholar). This domain acts as an autoinhibitory domain in the soluble form of the enzyme (10Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11Friesen J.A. Campbell H.A. Kent C. J. Biol. Chem. 1999; 274: 13384-13389Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). It is a mixed structure in the lipid-free enzyme, but upon membrane binding it adopts a purely α-helical conformation (12Taneva S. Johnson J.E. Cornell R.B. Biochemistry. 2003; 42: 11768-11776Crossref PubMed Scopus (41) Google Scholar). How does partitioning into a membrane bilayer relieve the inhibition at the active site? As part of our effort to answer this question we have examined changes in the quaternary interactions of the CCT dimer upon interaction with lipid vesicles. Early work established a homodimer structure for CCT by chemical cross-linking (13Cornell R. J. Biol. Chem. 1989; 264: 9077-9082Abstract Full Text PDF PubMed Google Scholar) and by gel filtration and sedimentation (14Weinhold P.A. Rounsifer M.E. Charles L. Feldman D.A. Biochim. Biophys. Acta. 1989; 1006: 299-310Crossref PubMed Scopus (41) Google Scholar). Craig et al. (8Craig L. Johnson J.E. Cornell R.B. J. Biol. Chem. 1994; 269: 3311-3317Abstract Full Text PDF PubMed Google Scholar) provided the first hint that the N-terminal two-thirds of the protein are sufficient for dimerization. After proteolytic digestion, which removed the more sensitive C-terminal domains, a species double the mass of the major N-terminal fragment could be detected on SDS-gels. The first x-ray structure of a cytidylyltransferase (glycerolphosphate cytidylyltransferase; GCT), which is homologous to the catalytic domain (amino acids 78–210) of CCT, showed that this fold interacts as a homodimer and gave insight into the dimer interface (15Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (89) Google Scholar). In particular, the Arg within one of three signature motifs for the cytidylyltransferase family, 139RYVDEVV145, is found at the dimer interface in GCT. A CCT truncated at residue 236, containing the N-terminal domain plus the entire catalytic domain, migrates as a dimer on gels after chemical cross-linking and in sedimentation analysis (11Friesen J.A. Campbell H.A. Kent C. J. Biol. Chem. 1999; 274: 13384-13389Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). This suggests that the dimerization of CCTα does not require the C-terminal region. There are no data from these papers specifying that the catalytic domain alone mediates dimerization. Other domains may participate in addition to the catalytic domain. For purposes of this work, the domains of CCT are designated domain N (1–72), domain C (73–236), domain M (237–300), and domain P (301–367) (Fig. 1). The boundaries are approximate. One goal of the present work was to map out which domains are involved in dimerization. This problem was approached using 1) chemical cross-linking of a limited protease digest of purified CCT followed by SDS-PAGE and antibody mapping to identify cross-linked species; 2) yeast two-hybrid interaction analysis of various CCT fragments; and 3) comparison of the disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. The results show that along with the catalytic domain the N-terminal domain (1–72) contributes to dimerization. The other goal was to examine whether the dimer interaction is modulated by membrane binding. We found that membrane binding perturbs the dimer interface, as detected by a loss of chemical cross-linking potential. This may reflect increased distance between contact sites or reduced flexibility at those sites. Materials—α-Chymotrypsin, PMSF, and phosphoglycerate mutase were from Sigma. Glutaraldehyde was from BDH. DSP and BS3 were from Pierce. [14C]NEM was from PerkinElmer Life Sciences. Alexa Fluor 532 succinimidyl ester, Prolong antifade, and Oregon Green- or Texas Red-conjugated secondary antibodies were from Molecular Probes. Lipids were from Avanti or Northern Lipids. Restriction enzymes, Vent polymerase, and T4 ligase were from New England Biolabs. Oligonucleotides were from Invitrogen. Antiserum against the N-terminal 15 residues of rat CCTα and against a 33-residue segment of domain M were obtained as described previously (16Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Sanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar, 17Johnson J.E. Aebersold R. Drobnies A. Cornell R.B. Biochim. Biophys. Acta. 1997; 1324: 273-284Crossref PubMed Scopus (39) Google Scholar). Antibody against amino acids 164–176 was generously provided by A. J. Ryan and R. K. Mallampalli (Iowa). Anti-hemagglutinin antibody was from Covance. The yeast two-hybrid vector pACT was from Clontech, and pBTM116 (18Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford, UK1993: 153Google Scholar) was a gift from Dr. Charles Boone (Toronto). SYPRO Orange protein gel stain was from Amersham Biosciences. Microcon-30 filters were from Amicon. Yeast Two-hybrid Construction and Analysis—Constructs were prepared which linked the LexA DNA binding domain (DBD) or the Gal4 activation domain (AD) to the N termini of full-length CCT or CCT fragments (amino acids 1–236, domains N + C; 73–239, domain C; 237–367, domains M + P). For this purpose we used the yeast expression vectors pBTM116 containing full-length LexA (18Bartel P.L. Chien C.-T. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practical Approach. Oxford University Press, Oxford, UK1993: 153Google Scholar), and pACT2 containing residues 768–881 of Gal4 and a nuclear targeting signal upstream of the Gal4 domain (24Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (299) Google Scholar). All CCT sequences were inserted in-frame into the BamHI or BamHI/SalI sites in the linker regions of these vectors. We engineered stop codons and BamHI or SalI sites flanking the CCT sequences by PCR mutagenesis, using WT rat CCTα in pAX142 (16Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Sanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar) as the template DNA. For CCT(1–367) and CCT(1–236) the 5′-primer was 5′-AT AGG ATC CCT TAT AAC ATG1 GAT2 GCA3 CAG4 AGT5 TCA6 GCT7 AAA8 GTC9-3′ (BamHI site in italics), adding a linker encoding RYN before the first CCT codon (CCT codons indicated with superscript). The 3′-primer for CCT(1–236) was 5′-ATA GGA TCCTCAstop GTT236 GAT235 AAA234 GCT233 GAC232 ATT231 GAG230 CTC229-3′. The 3′-primer for CCT(1–367) was ATA GGA TCCTCAstop GTC367 CTC366 TTC365 ATC364 CTC363 GCT362 GAT361 GTC360. These oligonucleotides introduced stop codons after codon 236 or 367 followed by the BamHI site. The CCT(237–367) construct was generated with the same 3′-primer as used to generate CCT(1–367), and the 5′-primer was 5′-AT AGG ATC CGT TAT AAC ATG GAA237 AAG238 AAA239 TAC240 CAC241 TTG242 CAA243 GAA244-3′, which added RYNM to the linker sequence just before CCT codon 237. CCT(73–239) was generated using a 5′-primer, 5′-AT AGG ATC CGT TAT AAC ATG TGT73 GAG74 CGG75 CCT76 GTG77 AGA78 GTT79-3′. The 3′ primer was 5′-T CGT CGA CTAstop TTT239 CTT238 TTC237 GTT236 G-3′, which introduced a SalI site flanking the stop codon following codon 239. The correct sequences were checked by inserting the amplified DNA into the BamHI or BamHI/SalI sites of pBS KS+ and sequencing both strands. For the pBTM and pACT CCT1–367 constructs a 1.1-kb 3′-EcoRI fragment was replaced with an EcoRI fragment from a previously sequenced full-length CCT so that only the 5′-end of the new construct required sequencing. To test interactions we used yeast strain Y274 containing the promoter sequence specific for LexA upstream of His-3 and LacZ, and lacking leucine and tryptophan synthases for selection of pBTM116 and pACT2 transformants. Yeast cells were made competent by modification of a lithium acetate procedure (24Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (299) Google Scholar) and were transformed using 33% polyethylene glycol and a 15-min, 42 °C heat shock (24Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (299) Google Scholar). Transformants were selected by culturing in leucine- and tryptophan-deficient medium at 30 °C for 48 h. To quantify the levels of LacZ expression, yeast cell lysates were prepared from mid-log phase cultures (A600 nm = 1.0). Cells from 3-ml cultures were collected by centrifugation at 13,000 rpm for 1 min and were suspended in 160 μl of Z buffer (60 mm Na2HPO4, 40 mm NaH2PO4, 10 mm KCl, 1 mm MgSO4) with 50 mm β-mercaptoethanol. 40 μl of sample was diluted to 1 ml with 0.15 m NaCl, 3.7% formaldehyde, and the A600 nm was determined. The remainder was solubilized by adding sarkosyl and toluene to final concentrations of 0.2%, and 3.8%, respectively. The sample was vortexed and centrifuged at 13,000 rpm for 10 s. The solvent was evaporated from the supernatant, and 100 μl was mixed with 650 μl of Z buffer containing 0.67 mg/ml o-nitrophenyl β-d-galactopyranoside to initiate the β-galactosidase reaction. The reaction was quenched at various times with 250 μl of 1 m Na2CO3 to a final concentration of 250 mm. The absorbance at 420 nm was determined. The units of β-galactosidase activity were calculated as shown in Equation 1. units=(A420 nm×40μl)/(A600 nm×100μl×time) (Eq. 1) Isolation of Yeast Spheroplasts and Nuclei and Analysis of Expression Levels of CCT Constructs—Cell walls were digested by zymolyase in the presence of 1 m sorbitol, essentially as described previously (25Randez-Gil F. Herrero P. Sanz P. Prieto J.A. Moreno F. FEBS Lett. 1998; 425: 475-478Crossref PubMed Scopus (70) Google Scholar). Preparation of spheroplasts was checked by microscopy. Nuclei were isolated from Dounce-homogenized spheroplasts by differential sedimentation in 18% Ficoll (25Randez-Gil F. Herrero P. Sanz P. Prieto J.A. Moreno F. FEBS Lett. 1998; 425: 475-478Crossref PubMed Scopus (70) Google Scholar). The nuclear prep was stained with DAPI and examined by microscopy using a Zeiss LSM-410 confocal microscope to assess the enrichment with nuclei. Immunoblotting was performed as described previously (26Northwood I.C. Tong A.H. Crawford B. Drobnies A.E. Cornell R.B. J. Biol. Chem. 1999; 274: 26240-26248Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) except that that transfer buffer was 39 mm glycine, 48 mm Tris, 0.0375% SDS, pH 8.7. For immunofluorescence analysis of CCT localization, spheroplasts were absorbed onto 0.1% polylysine-coated slides. The cells were fixed using sequential methanol and acetone baths at -20 °C, for 6 min and 30 s, respectively. After blocking with 2% bovine serum albumin in phosphate-buffered saline for 1 h, anti-hemagglutinin or anti-domain M antibodies were added in blocking buffer using a dilution of 1:100 for incubation over-night at 20 °C. After five washes, the secondary antibodies conjugated to Oregon Green or Texas Red were applied at 1:100 dilution in blocking buffer. The slides were prepared for viewing with Prolong antifade. The cells were viewed with a Zeiss LSM-410 microscope (26Northwood I.C. Tong A.H. Crawford B. Drobnies A.E. Cornell R.B. J. Biol. Chem. 1999; 274: 26240-26248Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Construction of Cys Mutants—WT CCTα was engineered with an N-terminal His-tag by PCR amplification using pBSKS(-) WT rat CCTα as the template. The forward primer, 5′-CAG CAG GTC TAG ACG CGT AGG ACC ATG GCT AAG CAC CAC CAT CAC CAT CAC ata gaa gga aga TCT GCC ATG1 GAT2 GCA3 CAG4 AGT5 TC-3′ introduced the His-tag (bold), a Factor Xa site (lowercase), and an optimal translation initiation sequence (underlined). Superscripts denote CCT codons. T3 was used as the reverse primer. The PCR product was inserted into pBSKS(-) at the XbaI and SalI sites. For expression in COS cells it was inserted in pAX142 at the MluI and SalI sites. The C139S was engineered by PCR using as template a His-tagged WT CCT in pBSKS(-) with a deleted SalI site. The forward primer was the His-tag-generating primer (see above), and the reverse primer introduced the mutation (underlined) at codon 139 followed by the AccI site (italics): 5′-CAC145 CTC144GTC143TAC142 GTA141 TCT140AGA139 ATG138 CTG137 CAC136 CGC135 GTC134-3′. The PCR product was inserted into pBS WT CCT with a deleted SalI site using MluI and AccI. For expression in COS cells it was inserted in pAX142 at the MluI and EcoRV sites. To construct CCT with serines at positions 354 and 359, WT CCT in pBSKS(-) was PCR amplified with the forward primer: 5′-CCA348 GCA349AGC350TTA351 TCC352AGA353TCT354 AAG355 GCT356 GTG357ACT358AGT359 GAC360 ATC361 AGC362 GAG363-3′, to introduce Cys to Ser mutations (underlined) as well as three new restriction sites: HindIII, BglII, and SpeI (italics). T3 was used as the reverse primer. His-tagged WT CCT was amplified using 5′-GA CTG CCG CGG GTT53 GAC54 TTT55 AGT56 AAG57-3′ as a forward primer and 5′-CCT353 AGA352 TAA351GCT350TGC349 TGG348 GGA347 GGA346-3′ as a reverse primer to create a HindIII site (italics). The HindIII to XhoI fragment of the C354,359S DNA was ligated with the SstI to HindIII fragment of the pBS His-tagged WT CCT amplification. The resulting SstI to XhoI fragment was then inserted into SstI/XhoI-cleaved pBSKS(-) His-tagged WT CCT. For expression in COS cells the construct was inserted into pAX142 using MluI and SalI sites. Further single cysteine to serine mutations were engineered using the QuikChange PCR mutagenesis materials (Stratagene) following the manufacturer's instructions. The PCR template was His-tagged WT CCTα in pBSKS(-). The complementary primer pairs replaced the cysteine codon with a serine codon. The sequences of the mutagenic codons of the forward primers were: C37S, TGT37 to TCT37; C68S, TGC68 to AGC68; C73S, TGT73 to TCT73; C113S, TGC113 to AGC113. The MluI to EcoRV fragments of the single cysteine mutants were each subcloned into MluI/EcoRV-cleaved pAX142-WT CCT. Cysteine-free CCT was engineered by replacing the N-terminal cysteines with serines one after another using the QuikChange PCR mutagenesis kit, with pBS-His-tagged C139S CCT as a template. MluI and EcoRV were used to subclone this into pAX142-HisC354,359S CCT. A single cysteine was engineered back into codon 37 using His-tagged Cys-free CCT in pBSKS(-) as a template. The sequence of the mutagenic codon of the forward primer was TCT37 to TGC37. All mutants were sequenced in pBSKS(-) to ensure correct sequence. Purification of Untagged CCT and Activity Assay—The α isoform of rat CCT was expressed in Trichoplusia ni cells using the baculovirus system (19MacDonald J.I. Kent C. Protein Expr. Purif. 1993; 4: 1-7Crossref PubMed Scopus (32) Google Scholar). It was purified by the method of Friesen et al. (11Friesen J.A. Campbell H.A. Kent C. J. Biol. Chem. 1999; 274: 13384-13389Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) with modifications (4Davies S.M. Epand R.M. Kraayenhof R. Cornell R.B. Biochemistry. 2001; 40: 10522-10531Crossref PubMed Scopus (110) Google Scholar). The purified CCT was stored in Buffer A (10 mm Tris, pH 7.4, 0.15 m NaCl, 1 mm EDTA, 2 mm dithiothreitol) at -80 °C. The CCT activity assay was carried out as described previously (20Sohal P.S. Cornell R.B. J. Biol. Chem. 1990; 265: 11746-11750Abstract Full Text PDF PubMed Google Scholar) in the presence of 0.1 mm PC/oleic acid (1:1)-sonicated vesicles. Expression and Purification of His-tagged CCTs—COS-1 cells were transfected for 68 h with the His-tagged CCT cDNA constructs in pAX142 (16Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Sanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar). The soluble fraction was prepared as described previously (27Johnson J.E. Xie M. Singh L.M. Egde R. Cornell R.B. J. Biol. Chem. 2003; 278: 514-522Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), except the homogenization buffer was 20 mm KH2PO4, pH 7.4, containing a full compliment of protease inhibitors. Prior to the high speed centrifugation, binding buffer was added from a 10× stock to a final concentration of 5 mm Na2HPO4, pH 8.0, 0.5 m NaCl, 15 mm imidazole. A 1/8 volume of 50% nickel-agarose beads was incubated with the soluble protein by rotation at 4 °C for 1 h. Beads were washed three times with 50 mm Na2HPO4, pH 8.0, 500 mm NaCl, 25 mm imidazole, 1% Nonidet P-40 and three times with 50 mm Na2HPO4, pH 8.0, 100 mm NaCl, 25 mm imidazole. His-tagged proteins were eluted with 50 mm Na2HPO4, pH 8.0, 100 mm NaCl, 350 mm imidazole. The CCT in the preparations purified by this method represented from 61 to 88% of the total protein in the samples, as assessed by densitometry of silver or SYPRO Orange-stained gels. The activities of the purified CCTs were determined as described above. α-Chymotrypsin Digestion—The reactions were carried out typically in a volume of 30 μl at 37 °C in a shaking water bath for various times using 1–4 μm CCT, a 1:150 weight ratio of chymotrypsin to CCT, and Buffer A with 0.15 mm Triton X-100. The reactions were initiated with chymotrypsin and were stopped with PMSF to a final concentration of 2 mm. A 10 mm PMSF solution in ∼90 °C water was prepared from a 0.5 m dimethyl sulfoxide stock immediately before the assay and was maintained at 37 °C. Some of the samples were prequenched with PMSF prior to chymotrypsin treatment. Other digested samples were subsequently reacted with glutaraldehyde within 2 min of adding PMSF (see below). Samples were analyzed by 12% SDS-PAGE. Cross-linking Reactions—Stocks of CuSO4 and phenanthrolene were mixed 1:3 immediately before initiating the reactions. For cross-linking with Cu(Phe)3 or DSP, the dithiothreitol and Tris were removed from the CCT preparation by dialysis against 20 mm K2HPO4, pH 7.4, 0.1 m NaCl, 0.15 mm Triton X-100 or by Microcon-30 filtration followed by three washes in the above buffer. For other reactions this step was not needed. The reactions were typically in a volume of 30 μl at 37 °C in a shaking water bath. The reaction buffer was 20 mm phosphate, pH 7.4, the Triton concentration was adjusted to be equivalent in all samples (generally 75 μm), and the concentrations of CCT (0.5–8 μm) and phospholipid vesicles were as specified in the figure legends. Small unilamellar vesicles were prepared by sonication as described previously (3Arnold R.S. Cornell R.B. Biochemistry. 1996; 35: 9917-9924Crossref PubMed Scopus (84) Google Scholar). The reactions were initiated with cross-linking reagent using the concentrations and incubation times specified in the figure legends. The glutaraldehyde reaction was quenched with 100 mm ethanolamine; the DSP reaction was quenched with 100 mm ammonium acetate, Cu(Phe)3 was quenched with 10 mm NEM, and BS3 was quenched with 0.1 m glycine, pH 6.5. Some samples were prequenched prior to the addition of cross-linker. Samples were analyzed by 10% SDS-PAGE. Labeling with [14C]NEM—The reaction with NEM was done in a volume of 20 μl and used 2.5 μm CCT (dithiothreitol-free), 225 μm [14C]NEM (specific activity, 39 Ci/mol) in 20 mm phosphate buffer, pH 7.4, 1 mm EDTA, 0.075 mm Triton X-100, with or without 500 μm PG vesicles. The sample was incubated with shaking for 30 min at 37 °C. The reaction was quenched by boiling for 3 min in Laemmli sample buffer containing 2% β-mercaptoethanol. The stained CCT bands were excised from the gel, treated with 50% H2O2 at 70 °C for 17 h, and the radioactivity determined by liquid scintillation counting. Gel Electrophoresis and Western Blot Analysis—Samples were boiled for 3 min in sample buffer prior to separation of components by SDS-PAGE using 10 or 12% acrylamide (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205463) Google Scholar). For Cu(Phe)3 and DSP cross-linked samples, the sample buffer did not contain β-mercaptoethanol. SYPRO Orange staining of gels was done as described by the manufacturer, and the gels were visualized on the Typhoon 9410 Variable Mode Imager with a 488 nm laser and a 580 nm filter. Silver staining of gels was done as described previously (22Poehling H.-M. Neuhoff V. Electrophoresis. 1981; 2: 141-147Crossref Scopus (175) Google Scholar). Immunoblots were done as described (23Veitch D.P. Gilham D. Cornell R.B. Eur. J. Biochem. 1998; 255: 227-234Crossref PubMed Scopus (46) Google Scholar) with an antibody directed against the N-terminal 15 amino acids (15Weber C.H. Park Y.S. Sanker S. Kent C. Ludwig M.L. Struct. Fold Des. 1999; 7: 1113-1124Abstract Full Text Full Text PDF Scopus (89) Google Scholar) or residues 256–288 of domain M (17Johnson J.E. Aebersold R. Drobnies A. Cornell R.B. Biochim. Biophys. Acta. 1997; 1324: 273-284Crossref PubMed Scopus (39) Google Scholar). Immunoblots with an antibody directed against residues 164–176 of domain C were done at 37 °C with both the primary antibody (anti-domain C), and the secondary antibody, goat anti-rabbit horseradish peroxidase, diluted 1:1,000 in 1% gelatin, 0.1% Tween 20 in Tris-buffered saline. Cross-linking Captures Intradimeric Interactions—In this work we used a variety of cross-linking reagents to probe the quaternary interactions of CCT. Glutaraldehyde is a relatively nonselective reagent that can form covalent bonds with heteroatoms on lysine, tyrosine, histidine, and cysteine side chains (28Habeeb A. Hiramoto R. Arch. Biochem. Biophys. 1968; 126: 16-26Crossref PubMed Scopus (398) Google Scholar). These reactive groups must approach within ∼7 Å to be cross-linked by the aldehydes. DSP and BS3 contain succinimidyl groups separated by ∼12 Å that react selectively with lysine amino groups. Cu(Phe)3 selectively oxidizes cysteine sulfhydryls. When lipid-free CCT was incubated with glutaraldehyde or the succinimidyl-based reagents, the 42-kDa monomer species observed on gels was converted to a diffuse molecular set between 80 and 110 kDa (Fig. 2, A and B, lanes 4–6; Fig. 3, A, lanes 2–5, and C, lanes 2–4). Autoxidation during the dialysis step preceding treatment with DSP or Cu(Phe)3 generated species at 84 kDa and ∼120 kDa in addition to the monomeric band (Fig. 2C, lanes 1–3). After incubation with Cu(Phe)3 the 84 and 120 kDa bands became predominant, and the 42 kDa band disappeared (Fig. 2C, lanes 4–6). The diffuse DSP-linked band at 80–110 kDa has been identified as heterogeneously linked homodimers by two-dimensional electrophoresis (9Dunne S.J. Cornell R.B. Johnson J.E. Glover N.R. Tracey A.S. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (89) Google Scholar). The identity of the 120 kDa disulfide-linked species has not been resolved. The predominance of this band varied from experiment to experiment (e.g. see Fig. 4C). In addition, higher order cross-linked oligomers were present in many samples as minor species (Fig. 2).Fig. 3PG vesicles diminish cross-linking efficiency.A, 0.39 μm CCT was preincubated 2 min with or without 300 μm PG vesicles. Reactions were initiated with 1 mm) BS3 and were quenched at the indicated times with 0.1 m glycine. B, fluorescence analysis of the Sypro Orange-stained gel in A, using Image Quant. ▴, samples without PG; ▪, samples with PG. C, 0.64 μm CCT was" @default.
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• W2021513697 date "2004-07-01" @default.
• W2021513697 modified "2023-10-01" @default.
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