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• W2065942389 abstract "Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp8, Tyr43, Trp100, Leu110, and Tyr261 in PotB and Trp46, Asp108, Glu169, Ser196, Asp198, and Asp199 in PotC were strongly involved in spermidine uptake and that Tyr160, Glu172, and Leu274 in PotB and Tyr19, Tyr88, Tyr148, Glu160, Leu195, and Tyr211 in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp8 in PotB was important for insertion of PotB and PotC into membranes. Tyr43, Trp100, and Leu110 in PotB and Trp46, Asp108, Ser196, and Asp198 in PotC were found to be involved in the interaction with PotD. Leu110 and Tyr261 in PotB and Asp108, Asp198, and Asp199 in PotC were involved in the recognition of spermidine, and Trp100 and Tyr261 in PotB and Asp108, Glu169, and Asp198 in PotC were involved in ATPase activity of PotA. Accordingly, Trp100 in PotB was involved in both PotD recognition and ATPase activity, Leu110 in PotB was involved in both PotD and spermidine recognition, and Tyr261 in PotB was involved in both spermidine recognition and ATPase activity. Asp108 and Asp198 in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity. Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp8, Tyr43, Trp100, Leu110, and Tyr261 in PotB and Trp46, Asp108, Glu169, Ser196, Asp198, and Asp199 in PotC were strongly involved in spermidine uptake and that Tyr160, Glu172, and Leu274 in PotB and Tyr19, Tyr88, Tyr148, Glu160, Leu195, and Tyr211 in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp8 in PotB was important for insertion of PotB and PotC into membranes. Tyr43, Trp100, and Leu110 in PotB and Trp46, Asp108, Ser196, and Asp198 in PotC were found to be involved in the interaction with PotD. Leu110 and Tyr261 in PotB and Asp108, Asp198, and Asp199 in PotC were involved in the recognition of spermidine, and Trp100 and Tyr261 in PotB and Asp108, Glu169, and Asp198 in PotC were involved in ATPase activity of PotA. Accordingly, Trp100 in PotB was involved in both PotD recognition and ATPase activity, Leu110 in PotB was involved in both PotD and spermidine recognition, and Tyr261 in PotB was involved in both spermidine recognition and ATPase activity. Asp108 and Asp198 in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity. Polyamines (putrescine, spermidine, and spermine) play important roles in cell proliferation and differentiation (1Cohen S.S. A Guide to Polyamines. Oxford University Press, New York1998Google Scholar, 2Wallace H.M. Fraser A.V. Hughes A. Biochem. J. 2003; 376: 1-14Crossref PubMed Scopus (758) Google Scholar, 3Igarashi K. Kashiwagi K. Int. J. Biochem. Cell Biol. 2010; 42: 39-51Crossref PubMed Scopus (561) Google Scholar), and cellular polyamine content is regulated by biosynthesis, degradation, and transport (4Pegg A.E. Cancer Res. 1988; 48: 759-774PubMed Google Scholar, 5Igarashi K. Kashiwagi K. Plant Physiol. Biochem. 2010; 48: 506-512Crossref PubMed Scopus (126) Google Scholar). With regard to transport, we characterized three polyamine transport systems in Escherichia coli (5Igarashi K. Kashiwagi K. Plant Physiol. Biochem. 2010; 48: 506-512Crossref PubMed Scopus (126) Google Scholar, 6Kashiwagi K. Kuraishi A. Tomitori H. Igarashi A. Nishimura K. Shirahata A. Igarashi K. J. Biol. Chem. 2000; 275: 36007-36012Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 7Kashiwagi K. Innami A. Zenda R. Tomitori H. Igarashi K. J. Biol. Chem. 2002; 277: 24212-24219Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Those were the spermidine-preferential and putrescine-specific uptake systems, which belong to the family of ATP-binding cassette transporters (8Eitinger, T., Rodionov, D. A., Grote, M., Schneider, E. (2010) FEMS Microbiol. Rev., in press.Google Scholar), and a protein, PotE, involved in the excretion of putrescine by a putrescine-ornithine antiporter activity. We also examined the properties of CadB, which is a cadaverine-lysine antiporter, and found that both PotE and CadB play important roles for cell growth at acidic pH (9Soksawatmaekhin W. Kuraishi A. Sakata K. Kashiwagi K. Igarashi K. Mol. Microbiol. 2004; 51: 1401-1412Crossref PubMed Scopus (146) Google Scholar). It has been also reported that a new putrescine uptake protein, PuuP, functions when putrescine is used as an energy source (10Kurihara S. Oda S. Kato K. Kim H.G. Koyanagi T. Kumagai H. Suzuki H. J. Biol. Chem. 2005; 280: 4602-4608Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Two uptake systems (spermidine-preferential, PotABCD, and putrescine-specific, PotFGHI) consist of a periplasmic substrate-binding protein (PotD or PotF), two transmembrane proteins (PotB and C or PotH and I), and a membrane-associated ATPase (PotA or PotG) (5Igarashi K. Kashiwagi K. Plant Physiol. Biochem. 2010; 48: 506-512Crossref PubMed Scopus (126) Google Scholar). Crystal structures of the two substrate-binding proteins (PotD and PotF) have been determined (11Sugiyama S. Vassylyev D.G. Matsushima M. Kashiwagi K. Igarashi K. Morikawa K. J. Biol. Chem. 1996; 271: 9519-9525Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12Vassylyev D.G. Tomitori H. Kashiwagi K. Morikawa K. Igarashi K. J. Biol. Chem. 1998; 273: 17604-17609Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Each consists of two domains with an alternating β-α-β topology, similar to other periplasmic binding proteins (13Vyas N.K. Vyas M.N. Quiocho F.A. Science. 1988; 242: 1290-1295Crossref PubMed Scopus (328) Google Scholar, 14Kang C.H. Shin W.C. Yamagata Y. Gokcen S. Ames G.F. Kim S.H. J. Biol. Chem. 1991; 266: 23893-23899Abstract Full Text PDF PubMed Google Scholar). The polyamine binding site lies in a cleft between the two domains as determined by crystallography and site-directed mutagenesis (11Sugiyama S. Vassylyev D.G. Matsushima M. Kashiwagi K. Igarashi K. Morikawa K. J. Biol. Chem. 1996; 271: 9519-9525Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12Vassylyev D.G. Tomitori H. Kashiwagi K. Morikawa K. Igarashi K. J. Biol. Chem. 1998; 273: 17604-17609Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15Kashiwagi K. Pistocchi R. Shibuya S. Sugiyama S. Morikawa K. Igarashi K. J. Biol. Chem. 1996; 271: 12205-12208Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We also purified a membrane-associated ATPase (PotA) of the spermidine-preferential uptake system, and some properties of PotA, including the existence of an ATP-binding site in the NH2 terminus, have been determined (16Kashiwagi K. Endo H. Kobayashi H. Takio K. Igarashi K. J. Biol. Chem. 1995; 270: 25377-25382Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The crystal structures of HisP (17Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar) and MalK (18Diederichs K. Diez J. Greller G. Müller C. Breed J. Schnell C. Vonrhein C. Boos W. Welte W. EMBO J. 2000; 19: 5951-5961Crossref PubMed Scopus (272) Google Scholar), membrane-associated ATPases of the histidine and maltose uptake systems, have been determined. The structure of HisP was very similar to that of the NH2-terminal domain of MalK (17Hung L.W. Wang I.X. Nikaido K. Liu P.Q. Ames G.F. Kim S.H. Nature. 1998; 396: 703-707Crossref PubMed Scopus (614) Google Scholar, 18Diederichs K. Diez J. Greller G. Müller C. Breed J. Schnell C. Vonrhein C. Boos W. Welte W. EMBO J. 2000; 19: 5951-5961Crossref PubMed Scopus (272) Google Scholar), but HisP (258 amino acid residues) was smaller than MalK (372 amino acid residues), and MalK had an extra domain in the COOH terminus. It has been reported that the COOH terminus of MalK was critical for negative regulation of the mal operon (19Kühnau S. Reyes M. Sievertsen A. Shuman H.A. Boos W. J. Bacteriol. 1991; 173: 2180-2186Crossref PubMed Google Scholar). Furthermore, it was found that a mutant (E306K) of the COOH terminus of MalK affects its ATPase activity, suggesting a role for this region in the ATPase activity (20Hunke S. Landmesser H. Schneider E. J. Bacteriol. 2000; 182: 1432-1436Crossref PubMed Scopus (23) Google Scholar). PotA (378 amino acid residues) was also expected to have an extra COOH-terminal domain, similar to that in MalK. We found that the NH2-terminal domain (residues 1–250) was involved in the recognition of ATP and in the interactions of PotA with a second PotA subunit and with PotB and PotC. The COOH-terminal domain (residues 251–378) of PotA contained a site that regulates ATPase activity and a site involved in the spermidine inhibition of ATPase activity (7Kashiwagi K. Innami A. Zenda R. Tomitori H. Igarashi K. J. Biol. Chem. 2002; 277: 24212-24219Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In this study, we tried to identify the amino acids of PotB and PotC involved in spermidine transport by random and site-directed mutagenesis and determined the functions of individual amino acid involved in spermidine transport. A polyamine-requiring mutant, E. coli MA261 (speB speC serA thr leu thi) (21Cunningham-Rundles S. Maas W.K. J. Bacteriol. 1975; 124: 791-799Crossref PubMed Google Scholar), was generously provided by W. K. Maas, New York University School of Medicine. E. coli MA261 potB::Km was prepared from E. coli MA261 as described previously (22Kashiwagi K. Miyamoto S. Nukui E. Kobayashi H. Igarashi K. J. Biol. Chem. 1993; 268: 19358-19363Abstract Full Text PDF PubMed Google Scholar) and was grown in medium A in the absence of polyamines (23Kashiwagi K. Hosokawa N. Furuchi T. Kobayashi H. Sasakawa C. Yoshikawa M. Igarashi K. J. Biol. Chem. 1990; 265: 20893-20897Abstract Full Text PDF PubMed Google Scholar). Another polyamine-requiring mutant, E. coli DR112 (speA speB thi) (24Linderoth N. Morris D.R. Biochem. Biophys. Res. Commun. 1983; 117: 616-622Crossref PubMed Scopus (10) Google Scholar), generously provided by D. R. Morris, University of Washington, was grown as described previously (25Igarashi K. Kashiwagi K. Hamasaki H. Miura A. Kakegawa T. Hirose S. Matsuzaki S. J. Bacteriol. 1986; 166: 128-134Crossref PubMed Scopus (152) Google Scholar). An ornithine deficiency in DR112 was achieved by the addition of 1 mg of arginine/ml to the medium. A proton-translocating ATPase mutant, JM105atp−, was prepared as described previously (7Kashiwagi K. Innami A. Zenda R. Tomitori H. Igarashi K. J. Biol. Chem. 2002; 277: 24212-24219Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and grown in an 18-amino acid-supplemented medium containing 1% glucose (26Kashiwagi K. Igarashi K. J. Bacteriol. 1988; 170: 3131-3135Crossref PubMed Google Scholar). Protease OmpT- and Lon-deficient mutant BL21(DE3) (27Gottesman S. Annu. Rev. Genet. 1996; 30: 465-506Crossref PubMed Scopus (598) Google Scholar) and a membrane-bound protease FtsH-deficient mutant AR3291 (28Ogura T. Inoue K. Tatsuta T. Suzaki T. Karata K. Young K. Su L.H. Fierke C.A. Jackman J.E. Raetz C.R. Coleman J. Tomoyasu T. Matsuzawa H. Mol. Microbiol. 1999; 31: 833-844Crossref PubMed Scopus (188) Google Scholar), kindly supplied by T. Ogura, Kumamoto University School of Medicine, were also grown in an 18-amino acid-supplemented medium containing 1% glucose (26Kashiwagi K. Igarashi K. J. Bacteriol. 1988; 170: 3131-3135Crossref PubMed Google Scholar). A low copy number plasmid pMW119 (Nippon Gene) (29Yamaguchi K. Masamune Y. Mol. Gen. Genet. 1985; 200: 362-367Crossref PubMed Scopus (47) Google Scholar) was digested with BamHI and HindIII. The termini were made blunt-ended with a Klenow fragment and religated, and as a result the BamHI- and HindIII-recognized sequence disappeared from the plasmid. Then, the plasmid was digested with KpnI and Cfr9I, and the 5.5-kbp KpnI and Cfr9I fragment from pPT104 (23Kashiwagi K. Hosokawa N. Furuchi T. Kobayashi H. Sasakawa C. Yoshikawa M. Igarashi K. J. Biol. Chem. 1990; 265: 20893-20897Abstract Full Text PDF PubMed Google Scholar) was inserted (pMWpotABCD, 9.7 kbp). The plasmid was further digested with KpnI and Bpu1102I, treated with T4 DNA polymerase, and religated. The pMWpotABCD thus obtained was 8.5 kbp and used for the experiment in this study. Transformation of E. coli cells with pMWpotABCD was carried out as described by Maniatis et al. (30Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 250-251Google Scholar). Appropriate antibiotics (100 μg/ml ampicillin and 50 μg/ml kanamycin) were added during the culture of E. coli-containing pMWpotABCD. Random mutagenesis was carried out using a PCR-based strategy (31Leng X.H. Manolson M.F. Forgac M. J. Biol. Chem. 1998; 273: 6717-6723Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). To obtain 1.2 kbp of mutated potB and potC genes, PCR was performed using 5′-GAGCAAGAGGGCACGCCGCG-3′ and 5′-CCGATAAGCGTACAAAACGTCGCCGAAAAC-3′ as the 5′- and 3′-primers for potB, and 5′-TATTGCCGGATCCCTGCTGGTGATGCTGCC-3′ and 5′-TCACGGGCATTGTCGGTCAAC-3′ as the 5′- and 3′-primers for potC. The first cycle was carried out in the presence of a 200 μm concentration each of three dNTPs and a 0.5 μm concentration of the fourth dNTP, with a 200 μm concentration of the fourth dNTP added in the subsequent 24 cycles. Four separate reaction mixtures (with dA, dC, dG, and dT, each at low concentrations in the first cycle) were combined, purified, and digested with XbaI and SalI for potB gene and with SalI and BamHI for potC gene. The digested fragments were inserted to the same restriction sites of pMWpotABCD. The mutated potABCD was transformed to E. coli MA261 potB::Km, and mutants were selected judging from low activity of spermidine uptake. Site-directed mutagenesis of Asp to Asn, Glu to Gln, and Trp and Tyr to Leu on potB and potC genes was carried out with the QuikChange Site-directed mutagenesis kit (Stratagene). A list of oligonucleotide primers for mutagenesis has not been included but is available from the authors upon request. Mutations were confirmed by DNA sequencing using a CEQ8000 DNA genetic analysis system (Beckman Coulter). Antibodies against PotA and PotD were prepared as described previously (32Furuchi T. Kashiwagi K. Kobayashi H. Igarashi K. J. Biol. Chem. 1991; 266: 20928-20933Abstract Full Text PDF PubMed Google Scholar). Antibodies against PotB and PotC were prepared according to the method of Posnett et al. (33Posnett D.N. McGrath H. Tam J.P. J. Biol. Chem. 1988; 263: 1719-1725Abstract Full Text PDF PubMed Google Scholar) using the multiple antigenic peptides LVYWRASRLLNKKVE, which corresponds to the PotB COOH-terminal peptide, and LIARDKTKGNTGDVK, which corresponds to the PotC COOH-terminal peptide. Rightside-out membrane vesicles were prepared from E. coli MA261 potB::Km/pMWpotABCD according to the procedure of Kaback (34Kaback H.R. Methods Enzymol. 1971; 22: 99-120Crossref Scopus (552) Google Scholar), except that the concentration of lysozyme was decreased from 500 to 50 μg/ml. For Western blot analysis of PotA, PotB, PotC, and PotD proteins, rightside-out membrane vesicles (10 μg of protein for PotA and PotD, and 30 μg of protein for PotB and PotC) were separated by SDS-PAGE (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) on a 12% acrylamide gel and transferred to a polyvinylidene fluoride membrane (Immobilon P; Millipore). The levels of four spermidine uptake proteins were detected with ECL Western blotting reagents (GE Healthcare), except that 0.2% Triton X-100 was used instead of 0.05% Tween 20 (36Nielsen P.J. Manchester K.L. Towbin H. Gordon J. Thomas G. J. Biol. Chem. 1982; 257: 12316-12321Abstract Full Text PDF PubMed Google Scholar) and quantified by a LAS-1000 plus luminescent image analyzer (Fuji Film). Spermidine uptake by intact cells (E. coli MA261 potB::Km/pMWpotABCD) was measured as described previously (37Kashiwagi K. Kobayashi H. Igarashi K. J. Bacteriol. 1986; 165: 972-977Crossref PubMed Google Scholar) using 50 μg of protein of intact cells and 10 μm [14C]spermidine as substrate. Incubation was carried out for 10 min at 30 °C. Rightside-out membrane vesicles from E. coli DR112/pACYCpotABC (22Kashiwagi K. Miyamoto S. Nukui E. Kobayashi H. Igarashi K. J. Biol. Chem. 1993; 268: 19358-19363Abstract Full Text PDF PubMed Google Scholar) were prepared as described above. PotD protein was purified as described previously (11Sugiyama S. Vassylyev D.G. Matsushima M. Kashiwagi K. Igarashi K. Morikawa K. J. Biol. Chem. 1996; 271: 9519-9525Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Spermidine uptake by rightside-out membrane vesicles was measured as described above using 100 μg of protein of membrane vesicles and various amounts of PotD protein as shown in the Fig. Inside-out membrane vesicles were prepared from E. coli JM105 atp−/pMWpotABCD by French press treatment of the E. coli cells suspended in 0.1 m potassium phosphate buffer, pH 6.6, and 10 mm EDTA according to the method of Houng et al. (38Houng H.S. Lynn A.R. Rosen B.P. J. Bacteriol. 1986; 168: 1040-1044Crossref PubMed Google Scholar). ATPase activity was measured by the method of Lill et al. (39Lill R. Cunningham K. Brundage L.A. Ito K. Oliver D. Wickner W. EMBO J. 1989; 8: 961-966Crossref PubMed Scopus (307) Google Scholar), except that the reaction mixture (0.1 ml) contained 50 mm Tris-HCl, pH 7.5, 50 mm KCl, 10 mm MgCl2, 0.5 mm [γ-32P]ATP (specific activity, 20–50 cpm/pmol), and 10 μg of protein of inside-out membrane vesicles. Protein content was determined by the method of Lowry et al. (40Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The reaction mixture (95 μl), containing buffer 1 (50 mm potassium phosphate buffer, pH 6.6, 50 mm Hepes-KOH, pH 7.6, 10 mm MgSO4, 20 mm ascorbic acid, and 10 μm phenazine methosulfate), 50 μg of protein of rightside-out membrane vesicles prepared from E. coli DR112/pACYCpotABC (22Kashiwagi K. Miyamoto S. Nukui E. Kobayashi H. Igarashi K. J. Biol. Chem. 1993; 268: 19358-19363Abstract Full Text PDF PubMed Google Scholar), and 1 μg of PotD, was preincubated at 30 °C for 5 min. The reaction was started by the addition of 5 μl of 200 μm spermidine. After incubation at 30 °C for 5 min, the reaction mixture was placed on the top of 20% sucrose in buffer 1 containing 10 μm spermidine, and centrifuged at 12,000 × g for 15 min. The level of PotD in the precipitate containing rightside-out membrane vesicles was detected by Western blotting after SDS-PAGE (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) as described above. DNA-dependent transcription-translation reactions (41Zubay G. Annu. Rev. Genet. 1973; 7: 267-287Crossref PubMed Scopus (463) Google Scholar) were carried out as described previously (42Higashi K. Kashiwagi K. Taniguchi S. Terui Y. Yamamoto K. Ishihama A. Igarashi K. J. Biol. Chem. 2006; 281: 9527-9537Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) with some modifications using an E. coli T7 S30 Extract System for Circular DNA (Promega). A reaction mixture (0.02 ml) contained 0.8 μg of pT7-BCD or pT7-BC (22Kashiwagi K. Miyamoto S. Nukui E. Kobayashi H. Igarashi K. J. Biol. Chem. 1993; 268: 19358-19363Abstract Full Text PDF PubMed Google Scholar), 240 kBq of [35S]methionine (27 TBq/mmol), 0.1 mm each 19 amino acids without methionine, 6 μl of T7 S30 extract, and 8 μl of the attached reaction mixture. After incubation at 37 °C for 60 min, 5 μl of 20 mm methionine and 1 ml of ice-cold 5% trichloroacetic acid were added to the reaction mixture. After removal of supernatant, the pellet was dissolved with 25 μl of sample buffer for SDS-PAGE (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) and boiled for 2 min. A 20-μl aliquot was used for 15% SDS-PAGE, and fluorography was performed according to the method of Laskey and Mills (43Laskey R.A. Mills A.D. Eur. J. Biochem. 1975; 56: 335-341Crossref PubMed Scopus (3026) Google Scholar). Radioactivity of labeled protein was quantified using a BAS-2000II imaging analyzer (Fuji Film). To determine which amino acids in PotB and PotC are involved in spermidine uptake, PotB and PotC mutants that influence spermidine uptake were isolated by random mutagenesis. In some mutants, it was found that there were two mutations. In that case, individual mutants were constructed, and spermidine uptake was measured. If the activity of the mutant was reduced to <30% or 30–60% of the control, the mutated amino acids were defined as having strong and moderate involvement, respectively, in the activity. E. coli MA261 potB::Km was transformed with the mutant potABCD, and spermidine uptake was measured. As shown in Fig. 1, Leu110 in PotB and Ser196 and Asp198 in PotC were strongly involved in spermidine uptake, and Leu274 in PotB and Leu195 and Tyr211 in PotC were moderately involved. Furthermore, Asp349 and Asp374 in PotA were strongly involved, and Val38 in PotD was moderately involved in spermidine uptake. It is known that polyamines are recognized by proteins through interactions with acidic and aromatic amino acid residues (11Sugiyama S. Vassylyev D.G. Matsushima M. Kashiwagi K. Igarashi K. Morikawa K. J. Biol. Chem. 1996; 271: 9519-9525Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 12Vassylyev D.G. Tomitori H. Kashiwagi K. Morikawa K. Igarashi K. J. Biol. Chem. 1998; 273: 17604-17609Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15Kashiwagi K. Pistocchi R. Shibuya S. Sugiyama S. Morikawa K. Igarashi K. J. Biol. Chem. 1996; 271: 12205-12208Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). There are 9 aspartic acid, 6 glutamic acid, 6 tryptophan, and 9 tyrosine residues in PotB, and 8 aspartic acid, 4 glutamic acid, 4 tryptophan, and 9 tyrosine residues in PotC. These residues were individually mutated to aspargine, glutamine, and leucine, respectively, and spermidine uptake was measured. As shown in Fig. 2, Trp8, Tyr43, and Trp100 in PotB and Trp46, Asp108, Glu169, Asp198, and Asp199 in PotC were strongly involved in spermidine uptake, and Tyr160 and Glu172 in PotB and Tyr19, Tyr88, Tyr148, Glu160, and Tyr211 in PotC were moderately involved. In contrast, mutation of Tyr261 to Leu in PotB enhanced spermidine uptake activity by 2.4-fold. When the hydropathy profile of the proteins was evaluated, it became apparent that PotB and PotC proteins contained six putative transmembrane-spanning segments linked by hydrophilic segments of variable length (32Furuchi T. Kashiwagi K. Kobayashi H. Igarashi K. J. Biol. Chem. 1991; 266: 20928-20933Abstract Full Text PDF PubMed Google Scholar). Most of the key amino acids identified by mutagenesis were located either on the cytoplasmic or the periplasmic sides, but not on the transmembrane segments (Fig. 3). The results suggest that interactions between PotA or PotD and the transmembrane proteins PotB and PotC occur on both surfaces of the membrane. We next examined the functions of 11 amino acids in PotB and PotC that are strongly involved in spermidine uptake. The association of PotA, PotB, PotC, and PotD in the membrane was studied by Western blot analysis using rightside-out membrane vesicles. As shown in Fig. 4, association of the four proteins was only disrupted by the Trp8 mutant in PotB. With the Trp8 mutant, the level of all four proteins, but especially PotB and PotC, on the membrane was low. The results suggest that Trp8 in PotB is important for the insertion of PotB and PotC into the membrane, so that association of PotA and PotD to the membrane was inhibited in the Trp8 mutant. To show the importance of Trp8 for the insertion of PotB and PotC to membrane, Trp8 was mutated to 10 other amino acids, consisting of hydrophilic, hydrophobic, acidic, and basic amino acids. In all mutants, association of PotA and PotD to membrane was strongly inhibited because the insertion of PotB and PotC to membrane was inhibited (Fig. 5A).FIGURE 5Levels of PotA, B, C, and D proteins in rightside-out membrane vesicles prepared from E. coli cells containing control PotB or Trp8 mutated PotB (A and B) and synthesis of control PotB and Trp8 mutated PotB (C). Western blot analysis was performed as described in the legend of Fig. 4 using rightside-out membrane vesicles prepared from E. coli MA261 potB::Km (A) or protease-deficient cells (B) containing pMWpotABCD or pMW-mutated potABCD. Synthesis of PotB (or Trp8 mutated PotB), PotC, and PotD (or NH2-terminal PotD) in a cell-free system was performed as described under “Experimental Procedures” (C). WT means normal PotB. Results were reproducible in duplicate determinations. Relative amount shown in A is the average of duplicate determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that Trp8 in PotB is important for the insertion of PotB and PotC into the membrane rather than increasing or accelerating degradation of PotB, possible degradation of the Trp8 mutant by the OmpT, Lon, and FtsH proteases was studied using mutants deficient in these proteases. The levels of PotA, B, C, and D on membranes in these mutants did not increase significantly, suggesting that the OmpT, Lon, and FtsH proteases do not influence the membrane expression of PotA, B, C, and D (Fig. 5B). It was determined whether synthesis of PotB and PotC is influenced by mutation of Trp8 in PotB in a cell-free protein synthetic system. As shown in Fig. 5C, synthesis of PotB and PotC was nearly equal in both wild type and Trp8-mutated PotB, and synthesis of PotB and PotC was very low compared with synthesis of PotD in both cell-free systems synthesizing normal and Trp8-mutated PotB. These results suggest that synthesis of PotB and PotC may be partially coupled with the insertion of PotB and PotC, and the insertion of PotB and PotC to membrane is inhibited in the Trp8-mutated PotB. The Km and Vmax values were then determined in the 10 mutants of PotB and PotC. As shown in Table 1, the Km value was greatly increased in PotB Leu110 and Tyr261 and PotC Asp108, Asp198, and Asp199 mutants, and Vmax was greatly decreased without significant change of the Km value in PotC Trp46, Glu169, and Ser196 mutants, suggesting that the former is involved in recognition of spermidine and the latter in recognition of the PotD or PotA protein.TABLE 1Km and Vmax values of spermidine uptake activity of PotABCD or PotABCD containing mutated PotB or PotCMutantKmVmaxμmnmol·min·mg proteinWT0.134 ± 0.028.61 ± 0.08PotB-Y43L0.351 ± 0.012.16 ± 0.09PotB-W100L0.150 ± 0.011.55 ± 0.03PotB-L110P>320NDaND, not determined.PotB-Y261L0.520 ± 0.0415.6 ± 0.38PotC-W46L0.102 ± 0.010.74 ± 0.10PotC-D108N>320NDPotC-E169Q0.120 ± 0.020.91 ± 0.01PotC-S196P0.126 ± 0.010.89 ± 0.12PotC-D198N>320NDPotC-D199N>320NDa ND, not determined. Open table in a new tab The ATPase activity of PotABC protein complex was determined in the 10 mutants using inside-out membrane vesicles. As shown in Fig. 6A, ATPase activity was greatly decreased in mutants of PotB Trp100 and PotC Asp108, Glu169, and Asp198. A decrease in both the affinity for spermidine and the ATPase activity was observed in mutants of PotC Asp108 and Asp198, suggesting that spermidine movement from PotD to cytoplasm and the structural change of PotB and PotC by PotA ATPase were coupled. At the Tyr261 PotB mutant, ATPase activity was greatly enhanced (Fig. 6B), although the Km value for spermidine increased (see Table 1). Enhancement of spermidine uptake by the PotB Tyr261 mutant is probably due to an increase in ATPase activity. Among 11 PotB and PotC mutants, the function of PotB Tyr43 and PotC Trp46 and Ser196 was not yet identified. These amino acid residues are located on the periplasmic side, and association of PotD with the membrane seems to be weak in these mutants compared with wild type PotB and PotC (see Fig. 4). Thus, association of PotD with the 10 mutants was examined by measuring spermidine uptake activity using rightside-out membrane vesicles with various amounts of PotD. As shown in Fig. 7, association of PotD protein with PotB Y43L and W100L and PotC W46L and S196P was weak compared with association with wild type PotB and PotC. Apparent Kd values of PotD binding to the PotABC complex were 0.7 μm (wild type), 3.4 μm (PotB Y43L), 3.7 μm (PotB W100L), 4.1 μm (PotC W46L), and 5.6 μm (PotC S196P). However, spermidine uptake activity of six other mutants was not recovered even if 10 μm PotD protein was added to the reaction mixture (Fig. 7). We next measured the direct binding of PotD to membrane vesicles, i.e. PotB and PotC proteins. As shown in Fig. 8A, PotD binding to membrane vesicles was dependent on the presence of both spermidine and ATP. It was confirmed that spermidine and ATP-dependent PotD binding to the membrane vesicles was weakened in the vesicles containing PotB Y43L and W100L and PotC W46L and S196P mutants. Furthermore, PotD binding was weakened in the presence of spermidine and ATP in vesicles containing PotC D108N mutant, but it was not significantly altered in vesicles containing PotC E169Q and D199N mutants. In the case of PotB L110P and PotC D198N, PotD binding in the absence of spermidine and ATP was greater than that to normal PotB and PotC. These results suggests that PotD may directly bind vesicles containing PotB L110P and PotC D198N mutants in the absence of spermidine and ATP, and release of PotD from these vesicles may be disturbed. The characteristics of the key amino acids are illustrated on a model of the PotABCD spermidine transporter (Fig. 9) following the model of maltose transport system (44Oldham M.L. Khare D. Quiocho F.A. Davidson A.L. Chen J. Nature. 2007; 450: 515-521Crossref PubMed Scopus (413) Google Scholar). Amino acid residues (PotB Tyr43 and Leu110 and PotC Trp46, Ser196, Asp198, and Asp199) involved in PotD binding and spermidine recognition during spermidine passage from PotD to cytoplasm are located mainly at the periplasmic side, and those involved in the insertion of PotB and PotC proteins and PotA ATPase (PotB Trp8 and Tyr261 and PotC Asp108 and Glu169) are located mainly at the cytoplasmic side. Amino acid residues involved in both PotD and spermidine recognition as well as PotA ATPase are located in both periplasmic (PotC Asp198) and cytoplasmic (PotC Asp108) sides. The results suggest that spermidine passage from PotD to cytoplasm and PotA ATPase are coupled each other. With regard to ATP-binding cassette transporters, the crystal structures of the maltose (44Oldham M.L. Khare D. Quiocho F.A. Davidson A.L. Chen J. Nature. 2007; 450: 515-521Crossref PubMed Scopus (413) Google Scholar) and molybdenum (45Hollenstein K. Frei D.C. Locher K.P. Nature. 2007; 446: 213-216Crossref PubMed Scopus (393) Google Scholar) transporters in complex with their binding proteins have been resolved. The structure of these two transporters is similar, and it is thought that the structural change for substrate influx is caused by ATPase activity (44Oldham M.L. Khare D. Quiocho F.A. Davidson A.L. Chen J. Nature. 2007; 450: 515-521Crossref PubMed Scopus (413) Google Scholar, 45Hollenstein K. Frei D.C. Locher K.P. Nature. 2007; 446: 213-216Crossref PubMed Scopus (393) Google Scholar, 46Oldham M.L. Davidson A.L. Chen J. Curr. Opin. Struct. Biol. 2008; 18: 726-733Crossref PubMed Scopus (198) Google Scholar). The spermidine uptake system comprising PotABCD is one of the ATP-binding cassette tranporters. The structure of PotD, the periplasmic substrate (spermidine)-binding protein, and some properties of PotA, the membrane-bound ATPase have been clarified (7Kashiwagi K. Innami A. Zenda R. Tomitori H. Igarashi K. J. Biol. Chem. 2002; 277: 24212-24219Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 11Sugiyama S. Vassylyev D.G. Matsushima M. Kashiwagi K. Igarashi K. Morikawa K. J. Biol. Chem. 1996; 271: 9519-9525Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 15Kashiwagi K. Pistocchi R. Shibuya S. Sugiyama S. Morikawa K. Igarashi K. J. Biol. Chem. 1996; 271: 12205-12208Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, the structure-function relationships of the membrane proteins PotB and PotC were studied using mutant PotB and PotC proteins. It was found that Trp8 located in the NH2 terminus of PotB protein is essential for insertion of PotB and PotC into the membrane. Trp cannot be replaced by any other amino acid. Because insertion of PotC was also inhibited by the PotB Trp8 mutant, association between PotB and PotC is probably important for insertion of both proteins. It has been reported that a Trp residue was necessary for correct trafficking of proteolipid protein 1 (PLP1) from the endoplasmic reticulum to plasma membrane in COS-7 cells (47Koizume S. Takizawa S. Fujita K. Aida N. Yamashita S. Miyagi Y. Osaka H. Neuroscience. 2006; 141: 1861-1869Crossref PubMed Scopus (13) Google Scholar). These results indicate that Trp on membrane protein plays important roles for correct arrangement of membrane proteins. Spermidine uptake by PotABCD may be analogous to maltose uptake by the maltose transport system (8Eitinger, T., Rodionov, D. A., Grote, M., Schneider, E. (2010) FEMS Microbiol. Rev., in press.Google Scholar, 44Oldham M.L. Khare D. Quiocho F.A. Davidson A.L. Chen J. Nature. 2007; 450: 515-521Crossref PubMed Scopus (413) Google Scholar, 46Oldham M.L. Davidson A.L. Chen J. Curr. Opin. Struct. Biol. 2008; 18: 726-733Crossref PubMed Scopus (198) Google Scholar). It has been reported that EAA loops present in the cytoplasmic loops of MalF and MalG, membrane proteins of the maltose transporter, are important for interaction with ATPase subunit, MalK (48Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar, 49Daus M.L. Grote M. Müller P. Doebber M. Herrmann A. Steinhoff H.J. Dassa E. Schneider E. J. Biol. Chem. 2007; 282: 22387-22396Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). An EAA motif also exists in the cytoplasmic loop of PotB and PotC (see Fig. 3). When Glu172 in PotB and Glu160 in PotC were mutated to Gln, the spermidine transport activity was decreased by about 50%. Thus, the EAA motif in PotB and PotC is important for interaction with PotA. It is thought that PotA dimers interact with PotB and PotC with different angles (see Fig. 9). Thus, the existence of Glu169 in PotC next to the EAA motif is probably important for the interaction of PotA with PotC. We also found that Tyr43 and Leu110 in PotB and Trp46 and Ser196 in PotC, which exist in the periplasmic loop, are important for the interaction with spermidine-binding protein, PotD. It was also found that Val38 and Phe46 in PotD are involved in spermidine uptake (see Fig. 1). The sequence motif at residues 46–54 (FTKETGIKV) of PotD (11Sugiyama S. Vassylyev D.G. Matsushima M. Kashiwagi K. Igarashi K. Morikawa K. J. Biol. Chem. 1996; 271: 9519-9525Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) is highly conserved with residues 53–61 (FEKDTGIKV) of the maltose-binding protein. The results suggest that this region, located at the loop between α1 and βB in PotD, may be important for the interaction with PotB and PotC. Amino acid residues involved in recognition of spermidine were defined as those that influence the Km value for spermidine. They were located either at the periplasmic side (Leu110 in PotB and Asp198 and Asp199 in PotC) or at the cytoplasmic side (Tyr261 in PotB and Asp108 in PotC). Amino acid residues at the periplasmic side may be involved in the release of spermidine from PotD, and those at the cytoplasmic side may be involved in the release of spermidine to the cytoplasm together with ADP (see Fig. 9). With a Tyr261 mutation to Leu in PotB, both spermidine uptake activity and ATPase activity of PotA increased by 2.4-fold, and the Km value for spermidine increased by 3.9-fold. These data support an idea that Tyr261 in PotB is involved in the release of spermidine to the cytoplasm by PotA ATPase. Amino acid residues affecting ATPase activity are also located at both the periplasmic side (Asp198 in PotC) and at cytoplasmic side (Tyr261 in PotB and Asp108 and Glu169 in PotC). A plausible model is one in which the structures of the transmembrane proteins (PotB and PotC) change as a consequence of ATP binding to PotA (see Fig. 9), analogous to the mechanism reported for the maltose uptake system (44Oldham M.L. Khare D. Quiocho F.A. Davidson A.L. Chen J. Nature. 2007; 450: 515-521Crossref PubMed Scopus (413) Google Scholar, 46Oldham M.L. Davidson A.L. Chen J. Curr. Opin. Struct. Biol. 2008; 18: 726-733Crossref PubMed Scopus (198) Google Scholar, 50Chen J. Sharma S. Quiocho F.A. Davidson A.L. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 1525-1530Crossref PubMed Scopus (175) Google Scholar, 51Grote M. Polyhach Y. Jeschke G. Steinhoff H.J. Schneider E. Bordignon E. J. Biol. Chem. 2009; 284: 17521-17526Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Asp108 and Asp198 in PotC were involved in both PotD and spermidine recognition as well as PotA ATPase, strongly suggesting that spermidine influx and ATPase are coupled. The results also indicate that these two amino acid residues are key amino acid residues for spermidine uptake by PotABCD because they are involved in all three functions. Unlike maltose binding on the equivalent proteins in the maltose transport system (44Oldham M.L. Khare D. Quiocho F.A. Davidson A.L. Chen J. Nature. 2007; 450: 515-521Crossref PubMed Scopus (413) Google Scholar), a spermidine recognition site on the transmembrane segments of PotB and PotC could not be identified. Thus, our experimental results support the idea that the cytoplasmic side of PotB and PotC is open for binding to the PotD-spermidine complex in the presence of ATP, and the periplasmic side of PotB and PotC is open for the release of spermidine together with hydrolysis of ATP. It has been reported that the NH2-terminal domain (residues 1–250) of PotA contains an active ATPase center and that the COOH-terminal domain (residues 251–378) contains a regulatory site for ATPase activity (7Kashiwagi K. Innami A. Zenda R. Tomitori H. Igarashi K. J. Biol. Chem. 2002; 277: 24212-24219Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). We found that Asp349 and Asp374 in the COOH-terminal domain are involved in spermidine uptake activity (see Fig. 1), indicating that these two aspartic acid residues are involved in regulation of ATPase activity of PotA. We thank Dr. K. Williams for help in preparing the manuscript and Drs. W. K. Maas, D. R. Morris, and T. Ogura for providing E. coli strains." @default.
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• W2065942389 title "Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity" @default.
• W2065942389 cites W1485191693 @default.
• W2065942389 cites W1492571006 @default.
• W2065942389 cites W1502544270 @default.
• W2065942389 cites W1532205945 @default.
• W2065942389 cites W1591736283 @default.
• W2065942389 cites W1652423080 @default.
• W2065942389 cites W1678396584 @default.
• W2065942389 cites W1775749144 @default.
• W2065942389 cites W1866587882 @default.
• W2065942389 cites W1880872626 @default.
• W2065942389 cites W1893548010 @default.
• W2065942389 cites W1927723646 @default.
• W2065942389 cites W1938522541 @default.
• W2065942389 cites W1946720111 @default.
• W2065942389 cites W1966541226 @default.
• W2065942389 cites W1974620743 @default.
• W2065942389 cites W1983176420 @default.
• W2065942389 cites W1986413605 @default.
• W2065942389 cites W1990883103 @default.
• W2065942389 cites W1994344581 @default.
• W2065942389 cites W1997195183 @default.
• W2065942389 cites W2008239057 @default.
• W2065942389 cites W2012742738 @default.
• W2065942389 cites W2035506170 @default.
• W2065942389 cites W2040295826 @default.
• W2065942389 cites W2040316448 @default.
• W2065942389 cites W2052146488 @default.
• W2065942389 cites W2052939596 @default.
• W2065942389 cites W2058689517 @default.
• W2065942389 cites W2063623087 @default.
• W2065942389 cites W2076300996 @default.
• W2065942389 cites W2076760898 @default.
• W2065942389 cites W2076861984 @default.
• W2065942389 cites W2080098971 @default.
• W2065942389 cites W2080223187 @default.
• W2065942389 cites W2087938597 @default.
• W2065942389 cites W2099196052 @default.
• W2065942389 cites W2100837269 @default.
• W2065942389 cites W2111688750 @default.
• W2065942389 cites W2128194288 @default.
• W2065942389 cites W2133913965 @default.
• W2065942389 cites W2140707634 @default.
• W2065942389 cites W2155446791 @default.
• W2065942389 cites W2156440952 @default.
• W2065942389 cites W2174331966 @default.
• W2065942389 cites W222557949 @default.
• W2065942389 cites W988977465 @default.
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