Targeted suppression of the ferroxidase and iron trafficking activities of the multicopper oxidase Fet3p from Saccharomyces cerevisiae
Tóm tắt
The Fet3 protein in Saccharomyces cerevisiae is a multicopper oxidase tethered to the outer surface of the yeast plasma membrane. Fet3p catalyzes the oxidation of Fe2+ to Fe3+; this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p. Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe2+, one of which is essential also to the coupling of the ferroxidase and uptake processes. The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively. The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed. The only marked kinetic defects measured in vitro were increases in K
M for Fe2+ exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants. These results suggest that these three residues contribute to the ferroxidase specificity site in Fet3p. In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in 59Fe uptake. For the Fet3p(E185D) mutant, K
M for iron was 300-fold greater than the wild-type K
M, while Fet3p(E185A) was completely inactive in support of iron uptake. In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane. These results suggest that E185 contributes to Fe2+ binding to Fet3p and to the subsequent trafficking of the Fe3+ produced to Ftr1p.
Tài liệu tham khảo
Kosman DJ (2003) Mol Microbiol 47:1185–1197
Martins LJ, Jensen LT, Simon JR, Keller GL, Winge DR (1998) J Biol Chem 273:23716–23721
Dancis A, Roman DG, Anderson GJ, Hinnebusch AG, Klausner RD (1992) Proc Natl Acad Sci USA 89:3869–3873
Georgatsou E, Alexandraki D (1999) Yeast 15:573–584
Dix DR, Bridgham JT, Broderius MA, Byersdorfer CA, Eide DJ (1994) J Biol Chem 269:26092–26099
Dix D, Bridgham J, Broderius M, Eide D (1997) J Biol Chem 272:11770–11777
Hassett R, Dix DR, Eide DJ, Kosman DJ (2000) Biochem J 351:477–484
de Silva DM, Askwith CC, Eide D, Kaplan J (1995) J Biol Chem 270:1098–1101
Stearman R, Yuan DS, Yamaguchi-Iwai Y, Klausner RD, Dancis A (1996) Science 271:1552–1557
Yuan DS, Dancis A, Klausner RD (1997) J Biol Chem 272:25787–25793
Harris ZL, Klomp LWJ, Gitlin JD (1998) Am J Clin Nutr 67:972S–977S
Vulpe CD, Kuo YM, Murphy TL, Cowley L, Askwith C, Libina N, Gitschier J, Anderson GJ (1999) Nat Genet 21:195–199
Frieden E, Osaki S (1974) Adv Exp Med Biol 48:235–265
Hassett RF, Yuan DS, Kosman DJ (1998) J Biol Chem 273:23274–23282
Solomon EI, Sundaram UM, Machonkin TE (1996) Chem Rev 96:2563–2605
Blackburn NJ, Ralle M, Hassett R, Kosman DJ (2000) Biochemistry 39:2316–2324
Machonkin TE, Quintanar L, Palmer AE, Hassett R, Severance S, Kosman DJ, Solomon EI (2001) J Am Chem Soc 123:5507–5517
Palmer AE, Quintanar L, Severance S, Wang T-Z, Kosman DJ, Solomon EI (2002) Biochemistry 41:6438–6448
Severance S, Kosman DJ (2003) J Biol Chem (in press)
Kosman DJ (2002) In: Valentine JS, Gralla E (eds) Advances in protein chemistry. Elsevier, New York, pp 221–269
Lindley P, Card G, Zaitseva I, Zaitsev V, Reinhammar B, Selin-Lindgren E, Yoshida K (1997) J Biol Inorg Chem 2:454–463
Askwith CC, Kaplan J (1998) J Biol Chem 273:22415–22419
Bonaccorsi di Patti MC, Felice MR, Camuti AP, Lania A, Musci G (2000) FEBS Lett 472:283–286
Bonaccorsi di Patti MC, Paronetto MP, Dolci V, Felice MR, Lania A, Musci G (2001) FEBS Lett 508:475–478
Yamaguchi-Iwai Y, Stearman R, Dancis A, Klausner RD (1996) EMBO J 15:3377–3384
Sikorski RS, Heiter P (1989) Genetics 122:19–27
Hassett R, Kosman DJ (1995) J Biol Chem 270:128–134
Bradford MM (1976) Anal Chem 72:248–254
Bonaccorsi di Patti MC, Pascarella S, Catalucci D, Calabrese L (1999) Prot Eng 12:895–897
de Silva D, Davis-Kaplan S, Fergestad J, Kaplan J (1997) J Biol Chem 272:14208–14213
Young SN, Curzon G (1972) Biochem J 129:273–283
Huber CT, Frieden E (1970) J Biol Chem 245:3973–3978
Harris ZL, Durley AP, Man TK, Gitlin JD (1999) Proc Natl Acad Sci USA 96:10812–10817
McKie AT, Marciani P, Rolfs A, Brennan K, Wehr K, Barrow D, Miret S, Bomford A, Peters TJ, Farzaneh F, Hediger MA, Hentze MW, Simpson RJ (2000) Mol Cell 5:299–309
Askwith C, Eide D, Van Ho A, Bernard PS, Li L, Davis-Kaplan S, Sipe DM, Kaplan J (1994) Cell 76:403–410
Zaitseva I, Zaitsev V, Card G, Moshkov K, Bax B, Ralph A, Lindley P (1996) J Biol Inorg Chem 1:15–23
Brown MA, Stenberg LM, Mauk AG (2002) FEBS Lett 520:8–12
Zaitsev VN, Zaitseva I, Papiz M, Lindley PF (1999) J Biol Inorg Chem 4:579–587
Zaric SD, Popovic DM, Knapp E-W (2000) Chem Eur J 6:3935–3942
Dougherty DA (1996) Science 271:163–168
Horovitz A, Serrano L, Fersht AR (1991) J Mol Biol 219:5–9
Huffman DL, O'Halloran TV (2001) Annu Rev Biochem 70:677–701
Rosenzweig AC (2001) Acc Chem Res 34:119–128
Huffman DL, O'Halloran TV (2000) J Biol Chem 275:18611–18614
Anderson KS (1999) Methods Enzymol 308:111–145
Chidambaram MV, Barnes G, Frieden E (1983) FEBS Lett 159:137–140