Abstract
Wild-type apocytochrome c and its hydrophobic segment deleted mutants, named Δ28–39, Δ72–86 and Δ28–29/72–86 were constructed, expressed and highly purified respectively. Insertion ability into phospholipid monolayer, inducing leakage of entrapped fluorescent dye fluorescein sulfonate (FS) from liposomes, and translocation across model membrane system showed that the wild-type apoprotein and Δ28–39 almost exhibited the same characteristics, while mutants with segment 72–86 deletion did not. Furthermore, CD spectra, intrinsic fluorescence emission spectra, and the accessibility of the protein to the fluorescence quenchers: KI, acrylamide and HB demonstrated that the segment 72–86 deletion has a significant effect on the conformational changes of apocytochrome c following its interaction with phospholipid. On the basis of these results it is postulated that the C-terminal hydrophobic segment 72–86 plays an important role in the translocation of apocytochrome c across membrane.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Hartl FU, Pfanner N, Nicholson DW: Mitochondria protein import. Biochim Biophys Acta 988: 1–45, 1989
Scarpulla RC, Agne KM, Ray W: Isolation and structure of a rat cytochrome c gene. J Biol Chem 256: 6480–6486, 1981
Zimmermann R, Henning B, Neupert W: Different transport pathways of individual precursor proteins in mitochondria. Eur J Biochem 116: 455–460, 1981
Stuart RA, Nicholson DW, Neupert W: Import of apocytochrome c into the mitochondrial intermembrane space along a cytochrome c1 sorting pathway. J Biol Chem 265: 20210–20219, 1990
Hault FU, Neupert W: Protein sorting to mitochondria: Evolutionary conservations of folding and assembly. Science 247: 930–938, 1990
Thomas S, Pfanner N, Neupert W: MOM19, an import receptor for mitochondrial precursor proteins. Cell 59: 1061–1070, 1989
Hakvoort TB, Sprinkle JR, Margoliash E: Reversible import of apocytochrome c into mitochondria. Proc Natl Acad Sci USA 87: 4996–5000, 1990
Dumont ME, Cardillo TS, Sherman F: Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae. Mol Cell Biol 11: 5487–5496, 1991
Dumont ME, Schlichter JB, Sherman F: CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c. Mol Cell Biol 13: 6442–6451, 1993
Mayer A, Neupert W, Lill R: Translocation of apocytochrome c across the outer membrane of mitochondria. J Biol Chem 270: 12390–12397, 1995
Rietveld A, Ponjee GA, de Kruijff B: Investigations on the insertion of the mitochondrial precursor protein apocytochrome c into model membranes. Biochim Biophys Acta 818: 398–409, 1985
Stuart RA, Nicholson DW, Neupert W: Early steps in mitochondrial protein import: Receptor functions can be substituted by the membrane insertion activity of apocytochrome c. Cell 60: 31–43, 1990
Zhou LX, Jordi W, de Kruijff B: Influence of heme and importance of the N-terminal part of the protein and physical state of model membranes for the apocytochrome c-lipid interaction. Biochim Biophys Acta 942: 115–124, 1988
Snel MM, de Kruijff B, Marsh D: Interaction of spin-labeled apocytochrome c and spin-labeled cytochrome c with negatively charged lipids studied by electron spin resonance. Biochemistry 33: 7146–7156, 1994
Tong JC, Zhu LQ, Yang FY: Cloning and high-level expression of chicken apocytochrome c gene in Escherichia coli. Biochemistry (USA) 36: 1187–1195, 1995
Yang FY, Wang XS, Tong JC: Study on the translocation of chicken heart apocytochrome c with different unfolded states. Biochem Mol Biol Int 30: 867–876, 1993
Szoka F, Papahadjopoulos D: Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation. Proc Natl Acad Sci USA 75: 4194–4198, 1978
Rouser G, Fleischer S, Yamamoto A. Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5: 494–496, 1970
Han XH, Sui SF, Yang FY: A mini-trough for the study of membrane insertion ability of proteins. Thin Solid Films 284/285: 789–792, 1996
Yang FY, Li NX, Wang XS: The relationship between apocytochrome c translocation and the N-terminal amphiphilic helix. Sci Bull 35: 135–138, 1990
Burger KN, Denel RA, de Kruijff B: Dynamin is membrane-active: Lipid insertion is induced by phosphoinositides and phosphatidic acid. Biochemistry 40: 12485–12493, 2000
Wang XS, Yang FY: Correlation between unfolded states of apocytochrome c and its ability to pass lipid bilayer. Science in China (series B) 24: 35–41, 1994
Rietveld A, Sijens P, de Kruijff B: Interaction of cytochrome c and its precursor apocytochrome c with various phospholipid. EMBO J 2: 907–913, 1983
Rankin SE, Watts A, Pinheiro TJ: Electrostatic and hydrophobic contributions to the folding mechanism of apocytochrome c driven by the interaction with lipid. Biochemistry 37: 12588–12595, 1998
De Jongh, HH J, de Kruijff B: The conformational changes of apocytochrome c upon binding to phospholipid vesicles and micells of phospholipid based detergents: A circular dichroism study. Biochim Biophys Acta 1029: 105–112, 1990
Yue JC, Tu YP, Pang SZ: The quenching of Trp of Ca2+-ATPase by HB. Chinese Sci Bull 40: 76–79, 1995
Rietveld A, Jordi W, de Kruijff B: Studies on the lipid dependency and mechanism of the translocation of the mitochondrial precursor protein apocytochrome c across model membrane. J Biol Chem 261: 3846–3856, 1986
Wang XS, Tong JC, Yang FY: The ability of apocytochrome c to pass lipid bilayer is relevant with its folding state. Biochem Mol Biol Int 30: 597–605, 1993
Henning B, Koehler H, Neupert W: Receptor sites involved in posttranslational transport of apocytochrome c into mitochondria: Specificity, affinity, and number of sites. Proc Natl Acad Sci USA 80: 4963–4967, 1983
Pilon M, Jordi W, de Kruijff B: Interactions of mitochondrial precursor protein apocytochrome c with phosphatidylserine in model membranes. A monolayer study. Biochim Biophys Acta 902: 207–216, 1987
Gorrissen H, Rietveld A, de Kruijff B: Apocytochrome c binding to negatively charged lipid dispersions studied by spin-labeled electron spin resonance. Biochemistry 25: 2904–2910, 1986
Jordi W, Zhou LX, de Kruijff B: The importance of the amino terminus of the mitochondrial precursor protein apocytochrome c for translocation across model membranes. J Biol Chem 264: 2292–2301, 1989
Sprinkle JA, Theodorus BM, Margoliash E: Amino acid sequence requirements for the association of apocytochrome c with mitochondria. Proc Natl Acad Sci USA 87: 5729–5733, 1990
Matsuura S, Arpin M, Hannum C: In vitro synthesis and posttranslational uptake of cytochrome c into isolated mitochondria: Role of a specific addressing signal in the apocytochrome. Proc Natl Acad Sci USA 78: 4368–4372, 1981
Stuart RA, Neupert W, Tropschug M: Deficiency in mRNA splicing in a cytochrome c mutant of Neurospora crassa: Importance of carboxy terminus for import of apocytochrome c into mitochondria. EMBO J 6: 2131–2137, 1987
Rights and permissions
About this article
Cite this article
Wang, X., Han, X., Jia, S. et al. Change of apocytocrhome c translocation across membrane in consequence of hydrophobic segment deletion. Mol Cell Biochem 233, 39–47 (2002). https://doi.org/10.1023/A:1015502800914
Issue Date:
DOI: https://doi.org/10.1023/A:1015502800914