FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2036358454> ?p ?o ?g. }

• W2036358454 endingPage "33130" @default.
• W2036358454 startingPage "33123" @default.
• W2036358454 abstract "MalK, the ATP-binding cassette component of the Escherichia coli maltodextrin transporter, has long been known to control negatively the activity of MalT, a transcriptional activator dedicated to the maltose regulon. By using a biochemical approach and the soluble form of MalK as a model substrate, we demonstrate that MalK alone inhibits transcription activation by MalT in a purified transcription system. The inhibitory effect observed in vitro is relieved by maltotriose and by two malT mutations and one malK mutation known to interfere with MalT repression by MalK in vivo. MalK interacts directly with the activator in the absence of maltotriose but not in the presence of maltotriose. Conversely, MalK inhibits maltotriose binding by MalT. Altogether, these data strongly suggest that MalK and maltotriose compete for MalT binding. Part, if not all, of the MalK-binding site is located on DT1, the N-terminal domain of MalT. All of these features indicate that MalK inhibits MalT by the same mechanism as two other proteins, MalY and Aes, that also act as negative effectors of MalT by antagonizing maltotriose binding by MalT. These results offer new insights into the mechanism by which gene regulation can be accomplished by the ATPase component of a bacterial ATP-binding cassette-type importer. MalK, the ATP-binding cassette component of the Escherichia coli maltodextrin transporter, has long been known to control negatively the activity of MalT, a transcriptional activator dedicated to the maltose regulon. By using a biochemical approach and the soluble form of MalK as a model substrate, we demonstrate that MalK alone inhibits transcription activation by MalT in a purified transcription system. The inhibitory effect observed in vitro is relieved by maltotriose and by two malT mutations and one malK mutation known to interfere with MalT repression by MalK in vivo. MalK interacts directly with the activator in the absence of maltotriose but not in the presence of maltotriose. Conversely, MalK inhibits maltotriose binding by MalT. Altogether, these data strongly suggest that MalK and maltotriose compete for MalT binding. Part, if not all, of the MalK-binding site is located on DT1, the N-terminal domain of MalT. All of these features indicate that MalK inhibits MalT by the same mechanism as two other proteins, MalY and Aes, that also act as negative effectors of MalT by antagonizing maltotriose binding by MalT. These results offer new insights into the mechanism by which gene regulation can be accomplished by the ATPase component of a bacterial ATP-binding cassette-type importer. Recent studies have revealed that bacterial transport systems can be directly involved in signal transduction pathways and play a key role in gene regulation by displaying a regulatory function that is tightly coupled to their transport activity. Based on the few examples of transport-based sensory systems that have been characterized in detail to date, the mechanisms of signal transduction differ widely, depending on the system. For instance, BglF, a phosphotransferase system enzyme II that catalyzes the import and phosphorylation of β-glucosides, phosphorylates and sequesters the BglG antiterminator in the absence of β-glucoside transport, and dephosphorylates and releases it when transport resumes (1Lopian L. Nussbaum-Shochat A. O'Day-Kerstein K. Wright A. Amster-Choder O. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7099-7104Crossref PubMed Scopus (41) Google Scholar). Active sugar transport triggers the recruitment of the Mlc repressor by the dephosphorylated glucose-specific phosphotransferase system enzyme II (PtsG), thereby derepressing the Mlc-controlled genes (for a review, see Ref. 2Plumbridge J. Curr. Opin. Microbiol. 2002; 5: 187-193Crossref PubMed Scopus (119) Google Scholar). In the case of the ferric citrate transport system, substrate binding to the receptor located in the outer membrane triggers the activation of a specific sigma factor via a complex signaling cascade (3Braun V. Mahren S. Ogierman M. Curr. Opin. Microbiol. 2003; 6: 173-180Crossref PubMed Scopus (123) Google Scholar).This paper deals with the roles that ATP-binding cassette transporters play in signaling pathways. The model system is the Escherichia coli maltodextrin transporter, whose ATP-binding cassette component, the MalK protein, down-regulates the activity of MalT, a transcriptional activator that controls the expression of the maltose regulon (4Boos W. Böhm A. Trends Genet. 2000; 16: 404-409Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). MalT control by MalK was first recognized with the finding that malK null mutants express constitutively the maltose regulon, whereas MalK overproduction blocks mal gene induction (5Hofnung M. Hatfield D. Schwartz M. J. Bacteriol. 1974; 117: 40-47Crossref PubMed Google Scholar, 6Reyes M. Shuman H.A. J. Bacteriol. 1988; 170: 4598-4602Crossref PubMed Google Scholar). The observation that the repression by MalK is relieved both by MalT overexpression and by a class of malT mutations (malTc) that confer constitutive expression of the maltose regulon suggested further that MalT is the target of the negative control exerted by MalK (6Reyes M. Shuman H.A. J. Bacteriol. 1988; 170: 4598-4602Crossref PubMed Google Scholar).The maltodextrin transport system is one of the best characterized ATP-binding cassette transporters, and its study has provided a wealth of information about the mechanism of coupling between transport and ATP hydrolysis (7Davidson A.L. Science. 2002; 296: 1038-1040Crossref PubMed Scopus (16) Google Scholar). The physiology of the regulatory function of MalK is also well understood. MalT repression by MalK ensures that the maltose regulon is not induced by endogenous maltotriose, the inducer of the system, in the absence of external maltodextrins (8Bukau B. Ehrmann M. Boos W. J. Bacteriol. 1986; 166: 884-891Crossref PubMed Google Scholar). The concentration of maltotriose of internal origin is indeed high enough to cause full expression of the regulon in a malK strain in minimal medium supplemented with glycerol (8Bukau B. Ehrmann M. Boos W. J. Bacteriol. 1986; 166: 884-891Crossref PubMed Google Scholar). The uninduced level of expression of the maltose regulon observed in a wild-type strain in the absence of maltose in the growth medium corresponds to the residual MalT activity that escapes repression by MalK. In contrast, the mechanism of MalT inhibition by MalK remains unclear. Some MalTc variants are known to display a higher affinity for maltotriose and to be less sensitive to repression by MalK, which suggests that MalK inhibits MalT by competing with maltotriose (6Reyes M. Shuman H.A. J. Bacteriol. 1988; 170: 4598-4602Crossref PubMed Google Scholar, 9Dardonville B. Raibaud O. J. Bacteriol. 1990; 172: 1846-1852Crossref PubMed Google Scholar). However direct evidence for this model is still lacking. Böhm et al. (10Böhm A. Diez J. Diederichs K. Welte W. Boos W. J. Biol. Chem. 2002; 277: 3708-3717Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) identified a surface determinant on the C-terminal domain of MalK that is specifically involved in MalT repression and might represent the MalT binding site. Panagiotidis et al. (11Panagiotidis C.H. Boos W. Shuman H.A. Mol. Microbiol. 1998; 30: 535-546Crossref PubMed Scopus (73) Google Scholar) observed a MalT-MalK interaction in vitro, but this MalT-MalK complex did not respond to signals known to relieve MalT repression by MalK in vivo. Also, how maltodextrin entrance is coupled to derepression remains elusive. On the basis of genetic data, Panagiotidis et al. (11Panagiotidis C.H. Boos W. Shuman H.A. Mol. Microbiol. 1998; 30: 535-546Crossref PubMed Scopus (73) Google Scholar) proposed that only the ATP-bound form of MalK, which is expected to predominate when the transporter is resting, is able to repress MalT.MalT is the archetype of a family of ∼100-kDa transcriptional activators found in prokaryotes (12De Schrijver A. De Mot R. Microbiology. 1999; 145: 1287Crossref PubMed Scopus (73) Google Scholar, 13Valdez F. González-Cerón G. Kieser H.M. Servín-González L. Microbiology. 1999; 145: 2365-2374Crossref PubMed Scopus (29) Google Scholar). Transcription activation by MalT involves MalT self-association, cooperative binding to an array of MalT sites located in the target promoters, and stimulation of open complex formation by the RNA polymerase (14Vidal-Ingigliardi D. Richet E. Raibaud O. J. Mol. Biol. 1991; 218: 323-334Crossref PubMed Scopus (54) Google Scholar, 15Danot O. Raibaud O. Mol. Microbiol. 1994; 14: 335-346Crossref PubMed Scopus (24) Google Scholar, 16Schreiber V. Richet E. J. Biol. Chem. 1999; 274: 33220-33226Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A complex interplay of signaling compounds and proteins controls the activity of the MalT protein (4Boos W. Böhm A. Trends Genet. 2000; 16: 404-409Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). MalT is active only in the presence of ATP and maltotriose, both of which are required for multimerization (16Schreiber V. Richet E. J. Biol. Chem. 1999; 274: 33220-33226Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In addition to MalK, two other proteins, namely MalY, a β C-S lyase, and Aes, an acyl esterase, down-regulate MalT activity (17Reidl J. Boos W. J. Bacteriol. 1991; 173: 4862-4876Crossref PubMed Google Scholar, 18Peist R. Koch A. Bolek P. Sewitz S. Kolbus T. Boos W. J. Bacteriol. 1997; 179: 7679-7686Crossref PubMed Google Scholar). Recent studies have revealed that both MalY and Aes interact directly with MalT and compete with maltotriose for MalT binding (19Schreiber V. Steegborn C. Clausen T. Boos W. Richet E. Mol. Microbiol. 2000; 35: 765-776Crossref PubMed Scopus (49) Google Scholar, 20Joly N. Danot O. Schlegel A. Boos W. Richet E. J. Biol. Chem. 2002; 277: 16606-16613Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Available data suggest that unliganded MalT is in equilibrium between an inactive monomeric form and an active monomeric form prone to self-association. The inactive form would be stabilized by MalY or Aes, whereas the active form would be stabilized by maltotriose. The requirement for ATP as a positive effector of MalT might reflect a role for the ATPase activity of MalT in the competition between positive and negative effectors. Indeed, both ATP and ADP can promote MalT multimerization or inhibitory complex formation, but ATP is more effective in driving MalT self-association, whereas ADP is more effective in promoting the formation of an inhibitory complex (16Schreiber V. Richet E. J. Biol. Chem. 1999; 274: 33220-33226Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 19Schreiber V. Steegborn C. Clausen T. Boos W. Richet E. Mol. Microbiol. 2000; 35: 765-776Crossref PubMed Scopus (49) Google Scholar, 20Joly N. Danot O. Schlegel A. Boos W. Richet E. J. Biol. Chem. 2002; 277: 16606-16613Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar).Structural studies have revealed that MalT is composed of four domains (21Danot O. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 435-440Crossref PubMed Scopus (31) Google Scholar). DT1 (residues 1-241) binds and hydrolyzes ATP (21Danot O. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 435-440Crossref PubMed Scopus (31) Google Scholar). It also binds MalY and Aes (20Joly N. Danot O. Schlegel A. Boos W. Richet E. J. Biol. Chem. 2002; 277: 16606-16613Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 22Schlegel A. Danot O. Richet E. Ferenci T. Boos W. J. Bacteriol. 2002; 184: 3069-3077Crossref PubMed Scopus (36) Google Scholar) whereas maltotriose is bound by DT3 (residues 437-806) (21Danot O. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 435-440Crossref PubMed Scopus (31) Google Scholar, 23Steegborn C. Danot O. Huber R. Clausen T. Structure. 2001; 9: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). DT4 (residues 807-901), which belongs to the LuxR-type family of DNA-binding domains, contains the DNA-binding site and most likely also carries a surface determinant contacting the RNA polymerase (24Danot O. Vidal-Ingigliardi D. Raibaud O. J. Mol. Biol. 1996; 262: 1-11Crossref PubMed Scopus (26) Google Scholar). The transition between the inactive and the active form of MalT involves DT1, DT2, and DT3, three domains that are specific to the MalT family of transcriptional activators and are thought to constitute a signal integration module (21Danot O. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 435-440Crossref PubMed Scopus (31) Google Scholar).To gain insight into the mechanism whereby MalK modulates MalT activity, we developed an in vitro system that reproduces MalT inhibition by MalK. The observation that MalK causes a Mal- phenotype when overproduced (6Reyes M. Shuman H.A. J. Bacteriol. 1988; 170: 4598-4602Crossref PubMed Google Scholar) suggested that free MalK, i.e. the MalK form that is not associated with the membrane components of the transporter (MalF and MalG), is able to inhibit MalT. We therefore used the soluble form of MalK as a model substrate. We show that MalK alone impedes transcription activation by MalT in a purified transcription system. Characterization of the repression process revealed that MalK inhibits MalT by forming a complex with the activator and by blocking inducer binding. Part, if not all, of the MalK-binding site is present on DT1.EXPERIMENTAL PROCEDURESStrains and Plasmids—E. coli BL21(λDE3) has been described by Studier et al. (25Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5987) Google Scholar). BL21(λDE3) ΔmalT220 contains a deletion of the entire malT gene (19Schreiber V. Steegborn C. Clausen T. Boos W. Richet E. Mol. Microbiol. 2000; 35: 765-776Crossref PubMed Scopus (49) Google Scholar). Plasmids pOM163 and pOM163-T38R are pET28b(+) (Novagen) derivatives producing DT1 or DT1-T38R with a Strep tag at their C terminus (DT1S) (20Joly N. Danot O. Schlegel A. Boos W. Richet E. J. Biol. Chem. 2002; 277: 16606-16613Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Plasmid pOM174 was obtained by amplifying the MalK-encoding fragment of pKN101 (obtained from K. Nikaïdo) by PCR using the oligonucleotides KU001 (5′-GCGGCGCCATGGGGACCCACGATCAGGTCGA-3′) and KD001 (5′-GGCCGCGAAGCTTCAGGCTTTGTGTGTTTTGT-3′), digesting it with NcoI and HindIII, and inserting it between the NcoI and HindIII sites of pET28b(+) followed by the insertion of a His tag encoding linker, HTAG1/HTAG2 (HTAG1, 5′-CATGCATCATCATCACCATCA-3′; HTAG2, 5′-CATGTGATGGTGATGATGATG-3′), in the NcoI site of the intermediate construct. The sequence of the construct was verified. The encoded polypeptide has a M(H)6MG extension at the MalK N terminus. The pOM174-G346S derivative was obtained by replacing the EcoRIHindIII fragment of pOM174 with the EcoRI-HindIII fragment of pAB204 (10Böhm A. Diez J. Diederichs K. Welte W. Boos W. J. Biol. Chem. 2002; 277: 3708-3717Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar).Chemicals—ADP containing <0.2% ATP was purchased from Roche Applied Science. [14C]maltotriose (900 mCi/mmol) was obtained from America Radiolabeled Chemicals.Proteins—RNA polymerase holoenzyme (Eσ70) was from Epicentre. Wild-type MalT, MalTc26, and MalT T38R proteins were purified in the presence of ATP as described by Danot and Raibaud (15Danot O. Raibaud O. Mol. Microbiol. 1994; 14: 335-346Crossref PubMed Scopus (24) Google Scholar), Schreiber et al. (19Schreiber V. Steegborn C. Clausen T. Boos W. Richet E. Mol. Microbiol. 2000; 35: 765-776Crossref PubMed Scopus (49) Google Scholar) and Joly et al. (20Joly N. Danot O. Schlegel A. Boos W. Richet E. J. Biol. Chem. 2002; 277: 16606-16613Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), respectively. ATP-free MalT was prepared by precipitating the purified protein with ammonium sulfate and filtering the resuspended material through a G-75 Sephadex column according to Schreiber and Richet (16Schreiber V. Richet E. J. Biol. Chem. 1999; 274: 33220-33226Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The concentration of MalT was measured as described (16Schreiber V. Richet E. J. Biol. Chem. 1999; 274: 33220-33226Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). ATP-free MalT was used throughout this work.The MalK protein, with a His tag at its amino terminus, was purified from strain BL21(λDE3) harboring pOM174. Bacteria were grown at 37 °C in 1.5 liters of Luria-Bertani medium (5 g of yeast extract, 10 g of tryptone, and 10 g of NaCl, adjusted to pH 7) in the presence of 50 μg/ml kanamycin, induced with 1 mm isopropyl thio-β-d-galactoside at A600 = 0.8, and further grown at 18 °C for 3 h. Cells were harvested by centrifugation, resuspended in buffer A (20 mm Tris-HCl (pH 7.7), 150 mm NaCl, and 5 mm MgCl2) to a final A600 = 100, frozen in liquid nitrogen, and stored at -70 °C. Cells were disrupted by two passages through a French press cell at 16,000 p.s.i. After centrifugation (30 min at 30,000 × g), the supernatant was loaded on a 6-ml Ni-NTA 1The abbreviations used are: Ni-NTA, nickel-nitrilotriacetic acid; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate. -agarose (Qiagen) column equilibrated with buffer A. The column was washed with 9 column volumes of buffer A plus 20 mm imidazole and 9 column volumes of buffer A plus 40 mm imidazole. The protein was eluted with buffer A plus 200 mm imidazole. The eluted protein was kept on ice, where it remained active for at least 8 days. Protein concentration was determined according to Lowry et al. (26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as a standard.ATPase Assay—His-tagged MalK was preincubated at 30 °C for 5 min in 19 μl of reaction mixture containing 36 mm HEPES-KOH (pH 8), 7 mm Tris-HCl (pH 7.7), 27 mm tripotassium citrate, 15 mm NaCl, 10 mm magnesium acetate, 20 mm imidazole, 1 mm dithiothreitol, and 210 μg·ml-1 acetylated bovine serum albumin (Sigma) before the addition of 1 μl of [γ-32P] ATP (1 mm, 0.01 Ci/mmol) and further incubation at 30 °C. The reaction was stopped by adding 2 μl of 0.25 m EDTA. Aliquots (2 μl) were spotted on polyethyleneimine cellulose plates (Schleicher and Schüll) and developed in 1 m formic acid and 0.5 m LiCl. The chromatograms were dried and scanned on a PhosphorImager.Abortive Initiation Assay—MalT was preincubated at 30 °C for 15 min in 18 μl of reaction mixture containing 40 mm HEPES-KOH (pH 8), 9 mm Tris-HCl (pH 7.7), 27 mm tripotassium citrate, 17 mm NaCl, 11 mm magnesium acetate, 0.11 mm EDTA, 1.1 mm dithiothreitol, 22 mm imidazole, 230 μg·ml-1 acetylated bovine serum albumin (Sigma), 5 nmmalPp DNA fragment, and the indicated concentrations of maltotriose, AMP-PNP, and MalK. Two microliters of RNA polymerase solution (0.13 μm in 40 mm HEPES-KOH (pH 8), 33 mm tripotassium citrate, 1 mm dithiothreitol, and 0.1 mg·ml-1 acetylated bovine serum albumin) was added, and the mixture was incubated for 15 min at 30 °C. The synthesis of abortive products (ApApC) was initiated by adding 2 μl of a solution containing 5 mm ApA, 0.5 mm [α-32P]CTP (0.2 Ci·mmol-1), and 500 μg·ml-1 heparin (H-0880; Sigma) and allowed to proceed for 15 min at 30 °C (heparin blocks open complex formation by trapping free RNA polymerase). The reaction products were separated from free [α-32P]CTP by chromatography on Whatman 3MM paper as described (27McClure W.R. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 5634-5638Crossref PubMed Scopus (270) Google Scholar). The chromatograms were dried and scanned on a Phosphor-Imager, and the amount of ApApC synthesized was quantified. The malPp DNA template is a 320-bp fragment containing the malPp promoter (from -154 to +130), prepared as described (19Schreiber V. Steegborn C. Clausen T. Boos W. Richet E. Mol. Microbiol. 2000; 35: 765-776Crossref PubMed Scopus (49) Google Scholar).Maltotriose Binding Assay—The 30-μl reaction mixture contained 40 mm HEPES-KOH (pH 8), 7 mm Tris-HCl (pH 7.7), 27 mm tripotassium citrate, 15 mm NaCl, 10 mm magnesium acetate, 0.1 mm EDTA, 1 mm dithiothreitol, 20 mm imidazole, 2 mm ATP, 0.5 μm [14C]maltotriose (900 mCi·mmol-1), 0.2 mg·ml-1 acetylated bovine serum albumin, and MalT and MalK as indicated. After 10 or 30 min of incubation at 20 °C, the tube was chilled on ice for 5 min, and the proteins were precipitated by adding 180 μl of a solution containing 3.1 m (NH4)2SO4, 40 mm HEPESKOH (pH 8), 33 mm tripotassium citrate, 10 mm magnesium acetate, 0.1 mm EDTA, 1 mm dithiothreitol, and 2 mm ATP. After 5 min on ice, the precipitate was collected by a 10-min centrifugation in a microcentrifuge at 4 °C, washed with 120 μl of the same solution, dissolved in 500 μl of 10 mm Tris-HCl (pH 8) and 1 mm EDTA, and counted in 5 ml of liquid scintillation mixture for 5 min. All values represent the average of assays performed in duplicate and are corrected for the background level (60 cpm) measured in the absence of MalT and MalK. The variations observed between two assays did not exceed 13%.Affinity Chromatography with Immobilized MalK—Bacteria over-producing His-tagged MalK+ or MalK-G346S were prepared as for MalK purification. A 10-ml cell suspension (A600 = 100) was thawed, disrupted in a French press cell (16,000 p.s.i.), and centrifuged (60 min at 20,000 × g) to collect the supernatant. Affinity chromatography was performed at 4 °C in Micro Biospin® Bio-Rad columns packed with 50 μl of Ni-NTA-agarose (Qiagen). Solutions were passed through the columns by spinning at 5 × g for 30 s in a bench top centrifuge. The columns were equilibrated with buffer A and loaded with 2 ml of soluble extract. The columns were washed with buffer A (5 × 100 μl) and buffer A plus 40 mm imidazole (5 × 100 μl). The MalK-loaded column was then equilibrated with buffer A plus 2 mm ATP ± 10 mm maltotriose (2 × 100 μl), and purified MalT (200 μl at 0.5 mg·ml-1 in buffer A plus 2 mm ATP (±10 mm maltotriose) was allowed to flow through the column. Unbound proteins were washed out with 5 × 100 μl of buffer A plus 40 mm imidazole plus 2 mm ATP (±10 mm maltotriose). His-tagged MalK was eluted with 4 × 100 μl of buffer A plus 500 mm imidazole. 100-μl fractions were collected starting from the MalT washing step and analyzed by 12% SDS-PAGE (acrylamide/bisacrylamide, 37.5:1).Affinity Chromatography with Immobilized DT1—Soluble extracts containing DT1S or DT1S-T38R were prepared from BL21(λDE3) ΔmalT220 harboring pOM163 or pOM163-T38R, as described (20Joly N. Danot O. Schlegel A. Boos W. Richet E. J. Biol. Chem. 2002; 277: 16606-16613Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Affinity chromatography was performed at 4 °C in Micro Biospin® Bio-Rad columns packed with 50 μl of Strep-Tactin®-Sepharose® resin (IBA), as described above. The columns were equilibrated with buffer B (50 mm Tris-HCl (pH 7.7), 10% sucrose, 10 mm MgCl2, and 2 mm AMP-PNP) plus 0.5 m KCl and loaded with 250 μl of DT1S or DT1ST38R soluble extract supplemented with 2 mm AMP-PNP. The columns were washed with 6× 100 μl of buffer B plus 0.1 m KCl. Freshly purified MalK (1 ml at 0.25 mg/ml in the washing buffer) was allowed to flow through the DT1-loaded column, and unbound proteins were washed out with 5× 100 μl of the washing buffer. DT1S or DT1S-T38R was eluted with 4× 100 μl of buffer B plus 0.1 m KCl plus 2.5 mm desthiobiotin (Sigma). Ten 100-μl fractions were collected, starting with the MalK washing step, and analyzed by 12% SDS-PAGE (acrylamide/bisacrylamide, 37.5:1).RESULTSMalK Inhibits Transcription Activation by MalT in Vitro—We first examined whether purified MalK inhibits MalT in an in vitro transcription system. MalT activity was assayed by monitoring its ability to activate open complex formation at malPp, a MalT-dependent promoter, in the presence of RNA polymerase. MalT was incubated with malPp DNA and AMPPNP 2AMP-PNP, which can replace ATP as an effector of MalT (33Richet E. Raibaud O. EMBO J. 1989; 8: 981-987Crossref PubMed Scopus (83) Google Scholar), is actually more effective than ATP in driving MalT self-association in the presence of maltotriose due to the absence of ATP hydrolysis, which generates ADP-bound forms that are less prone to multimerization (16Schreiber V. Richet E. J. Biol. Chem. 1999; 274: 33220-33226Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). in the presence or absence of MalK before adding RNA polymerase and allowing open complex formation. The amount of the open complexes formed was then determined by measuring the rate of abortive product synthesis. The assay was performed in the presence of AMP-PNP instead of ATP to avoid MalK- and MalT-driven ATP hydrolysis, which might interfere with the assay. In addition, given that MalT-dependence on maltotriose is only partial in the presence of AMP-PNP, 3MalT is 50% as active in the presence of AMP-PNP alone as it is in the presence of AMP-PNP and a saturating concentration of maltotriose. N. Joly, unpublished results. maltotriose was omitted from the assay. This omission avoided the possibility that maltotriose might impede repression by MalK. Note also that, for a given concentration of MalT, the fraction of the protein that is in the active form, as judged by the promoter response, depends on the concentrations of the positive effectors and on the MalT variant assayed. Therefore, for each combination of effectors and MalT variant used in this work, we determined an individual response curve, i.e. the amount of open complexes formed as a function of MalT concentration. The concentration of MalT that was used to test the effect of MalK was that eliciting about half of the maximum response under the chosen conditions. The presence of a limiting concentration of MalT in the assay ensures that MalT inhibition by MalK can be readily detected.As shown in Fig. 1, MalK strongly depressed malPp activation by MalT in the absence of maltotriose; the amount of open complexes formed was reduced by 80% in the presence of 8 μm MalK. Furthermore, the inhibitory effect of MalK disappeared when the assay was performed in the presence of 0.1 mm maltotriose (Fig. 1). The fact that maltotriose antagonizes MalT inhibition by MalK, as was expected based on in vivo data, provides evidence that the inhibitory effect observed in vitro is physiologically relevant.malT and malK Mutations Relieving MalT Inhibition by MalK in Vivo Also Suppress the Inhibitory Effect of MalK in Vitro—To confirm that the inhibition caused by MalK in vitro is functionally significant, we examined whether two different malT mutations that are known to confer resistance to MalK in vivo (malTc26 and malT-T38R) have the same effect in vitro. The malTc26 mutation, which generates the R242P substitution in the DT1-DT2 linker, increases the affinity of MalT for maltotriose by favoring the transition from the inactive state to the active state (9Dardonville B. Raibaud O. J. Bacteriol. 1990; 172: 1846-1852Crossref PubMed Google Scholar, 21Danot O. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 435-440Crossref PubMed Scopus (31) Google Scholar). It also suppresses MalT-sensitivity to MalK in vivo (22Schlegel A. Danot O. Richet E. Ferenci T. Boos W. J. Bacteriol. 2002; 184: 3069-3077Crossref PubMed Scopus (36) Google Scholar). The T38R substitution, which is located in DT1, diminishes MalT-sensitivity to MalK in vivo (22Schlegel A. Danot O. Richet E. Ferenci T. Boos W. J. Bacteriol. 2002; 184: 3069-3077Crossref PubMed Scopus (36) Google Scholar). As shown in Fig. 2A, neither of these MalT variants was affected by MalK when assayed in the presence of AMP-PNP alone, i.e. under conditions in which wild-type MalT is inhibited by MalK.Fig. 2Effect of malT and malK mutations on MalT repression by MalK. Abortive initiation assays were performed in the presence of 0.5 mm AMP-PNP and the indicated concentrations of MalT and MalK variants. A, closed squares, 240 nm MalTc26; open diamonds, 300 nm MalT-T38R. B, closed squares, 240 nm wild-type MalT ± wild-type MalK; open diamonds, 240 nm wild-type MalT ± MalK-G346S. wt, wild-type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also tested whether the inhibitory effect associated with purified MalK was affected by the malK-G346S mutation. The G346S substitution, which is thought to alter the MalT-binding site, specifically decreases the ability of MalK to down-regulate MalT in vivo without damaging its ability to catalyze maltose transport both when overproduced or when constitutively expressed from a chromosomal locus (10Böhm A. Diez J. Diederichs K. Welte W. Boos W. J. Biol. Chem. 2002; 277: 3708-3717Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). 4E. Richet, unpublished results. As expected, MalT is not inhibited by the MalK-G346S variant in the presence of AMP-PNP alone (Fig. 2B). To rule out the possibility that the lack of effect of MalK-G346S was caused by protein misfolding, we verified that the purified MalK variant hydrolyzes ATP in vitro at exactly the same rate as the wild-type protein (0.32 and 0.33 molecule of ATP are hydrolyzed at 30 °C per minute per protomer of wild-type and mutant protein, respectively). The uncoupled ATPase activity of MalK is a good indicator of correct protein folding, given that ATP hydrolysis by MalK relies on protein dimerization and that dimerization involves both the nucleotide-binding domain and the regulatory domain, as revealed by the x-ray struct" @default.
• W2036358454 created "2016-06-24" @default.
• W2036358454 creator A5004902921 @default.
• W2036358454 creator A5031419306 @default.
• W2036358454 creator A5075851860 @default.
• W2036358454 creator A5080442262 @default.
• W2036358454 date "2004-08-01" @default.
• W2036358454 modified "2023-09-30" @default.
• W2036358454 title "MalK, the ATP-binding Cassette Component of the Escherichia coli Maltodextrin Transporter, Inhibits the Transcriptional Activator MalT by Antagonizing Inducer Binding" @default.
• W2036358454 cites W1499701561 @default.
• W2036358454 cites W1504211443 @default.
• W2036358454 cites W1586864304 @default.
• W2036358454 cites W1594720150 @default.
• W2036358454 cites W1598145342 @default.
• W2036358454 cites W1775749144 @default.
• W2036358454 cites W1821704317 @default.
• W2036358454 cites W1858736637 @default.
• W2036358454 cites W1873199501 @default.
• W2036358454 cites W1980879384 @default.
• W2036358454 cites W1985744441 @default.
• W2036358454 cites W2006373328 @default.
• W2036358454 cites W2009929728 @default.
• W2036358454 cites W2010363628 @default.
• W2036358454 cites W2026984793 @default.
• W2036358454 cites W2028754843 @default.
• W2036358454 cites W2039243578 @default.
• W2036358454 cites W2042294209 @default.
• W2036358454 cites W2048300537 @default.
• W2036358454 cites W2057011513 @default.
• W2036358454 cites W2058515102 @default.
• W2036358454 cites W2076705929 @default.
• W2036358454 cites W2088693912 @default.
• W2036358454 cites W2089778735 @default.
• W2036358454 cites W2099976574 @default.
• W2036358454 cites W2103414965 @default.
• W2036358454 cites W2115412292 @default.
• W2036358454 cites W2124530235 @default.
• W2036358454 cites W2124635010 @default.
• W2036358454 cites W2151917326 @default.
• W2036358454 cites W2156342386 @default.
• W2036358454 cites W2170289074 @default.
• W2036358454 cites W46142366 @default.
• W2036358454 doi "https://doi.org/10.1074/jbc.m403615200" @default.
• W2036358454 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15180985" @default.
• W2036358454 hasPublicationYear "2004" @default.
• W2036358454 type Work @default.
• W2036358454 sameAs 2036358454 @default.
• W2036358454 citedByCount "54" @default.
• W2036358454 countsByYear W20363584542012 @default.
• W2036358454 countsByYear W20363584542013 @default.
• W2036358454 countsByYear W20363584542014 @default.
• W2036358454 countsByYear W20363584542015 @default.
• W2036358454 countsByYear W20363584542017 @default.
• W2036358454 countsByYear W20363584542018 @default.
• W2036358454 countsByYear W20363584542019 @default.
• W2036358454 countsByYear W20363584542020 @default.
• W2036358454 countsByYear W20363584542023 @default.
• W2036358454 crossrefType "journal-article" @default.
• W2036358454 hasAuthorship W2036358454A5004902921 @default.
• W2036358454 hasAuthorship W2036358454A5031419306 @default.
• W2036358454 hasAuthorship W2036358454A5075851860 @default.
• W2036358454 hasAuthorship W2036358454A5080442262 @default.
• W2036358454 hasConcept C104317684 @default.
• W2036358454 hasConcept C149011108 @default.
• W2036358454 hasConcept C185592680 @default.
• W2036358454 hasConcept C2225953 @default.
• W2036358454 hasConcept C44312359 @default.
• W2036358454 hasConcept C547475151 @default.
• W2036358454 hasConcept C55493867 @default.
• W2036358454 hasConcept C86803240 @default.
• W2036358454 hasConcept C88045685 @default.
• W2036358454 hasConceptScore W2036358454C104317684 @default.
• W2036358454 hasConceptScore W2036358454C149011108 @default.
• W2036358454 hasConceptScore W2036358454C185592680 @default.
• W2036358454 hasConceptScore W2036358454C2225953 @default.
• W2036358454 hasConceptScore W2036358454C44312359 @default.
• W2036358454 hasConceptScore W2036358454C547475151 @default.
• W2036358454 hasConceptScore W2036358454C55493867 @default.
• W2036358454 hasConceptScore W2036358454C86803240 @default.
• W2036358454 hasConceptScore W2036358454C88045685 @default.
• W2036358454 hasIssue "32" @default.
• W2036358454 hasLocation W20363584541 @default.
• W2036358454 hasOpenAccess W2036358454 @default.
• W2036358454 hasPrimaryLocation W20363584541 @default.
• W2036358454 hasRelatedWork W2036358454 @default.
• W2036358454 hasRelatedWork W2052869590 @default.
• W2036358454 hasRelatedWork W2092246493 @default.
• W2036358454 hasRelatedWork W2099997130 @default.
• W2036358454 hasRelatedWork W2101822540 @default.
• W2036358454 hasRelatedWork W2340363796 @default.
• W2036358454 hasRelatedWork W2355938210 @default.
• W2036358454 hasRelatedWork W2770027949 @default.
• W2036358454 hasRelatedWork W3119662195 @default.
• W2036358454 hasRelatedWork W4294921690 @default.
• W2036358454 hasVolume "279" @default.
• W2036358454 isParatext "false" @default.
• W2036358454 isRetracted "false" @default.
• W2036358454 magId "2036358454" @default.

• next