http://www.cnr.it/ontology/cnr/individuo/prodotto/ID12708
Site-directed mutagenesis and chemical modification of the six native cysteine residues of the rat mitochondrial carnitine carrier: implications for the role of cysteine-136 (Articolo in rivista)
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- Label
- Site-directed mutagenesis and chemical modification of the six native cysteine residues of the rat mitochondrial carnitine carrier: implications for the role of cysteine-136 (Articolo in rivista) (literal)
- Anno
- 2002-01-01T00:00:00+01:00 (literal)
- Alternative label
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- Indiveri C; Giangregorio N; Iacobazzi V; Palmieri F (literal)
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- By use of site-directed mutagenesis in combination with chemical
modification of mutated proteins, the role of the six Cys residues in the
transport function of the rat mitochondrial carnitine carrier (CAC) was
studied. Several CAC mutants, in which one or more Cys residues had been
replaced with Ser, were overexpressed in Escherichia coli, purified, and
reconstituted in liposomes.Single, double, and quadruple replacement mutants showed V(max) comparable to that of the wild type. On the basis of the values of internal and external transport affinities (K(m)) for carnitine and of their comparison with those measured in mitochondria, the recombinant CAC is oriented unidirectionally in the liposomes, right side out compared to mitochondria.The wild-type
CAC and the mutants containing Cys-136 showed substrate protection against
NEM and MTSES inhibition and against NEM labeling. The data show that none
of the native cysteines is essential for the transport mechanism and that
Cys-136 is the major target of SH reagents and raise the hypothesis that
Cys-136 is accessible from the external medium and is located at, or near,
the substrate binding site. A model of the CAC is proposed in which the
matrix hydrophilic loop containing Cys-136 protrudes into the membrane
between the transmembrane domains of the protein.
(literal)
- Note
- ISI Web of Science (WOS) (literal)
- Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#affiliazioni
- Department of Cellular Biology, University of Calabria, Via P.Bucci cubo 4c, 87036 Arcavacata di Rende (CS), Italy
National Research Council Institute of Biomembranes and Bioenergetics (IBBE), via Amendola 165/A, 70126, Bari, Italy
Laboratory of Biochemistry and Molecular Biology, Department of Pharmaco-Biology, University of Bari, Via Orabona 4, 70125 Bari, Italy (literal)
- Titolo
- Site-directed mutagenesis and chemical modification of the six native cysteine residues of the rat mitochondrial carnitine carrier: implications for the role of cysteine-136 (literal)
- Abstract
- By use of site-directed mutagenesis in combination with chemical
modification of mutated proteins, the role of the six Cys residues in the
transport function of the rat mitochondrial carnitine carrier (CAC) was
studied. Several CAC mutants, in which one or more Cys residues had been
replaced with Ser, were overexpressed in Escherichia coli, purified, and
reconstituted in liposomes. The efficiency of incorporation into liposomes
of the reconstituted proteins was lower for all constructs lacking Cys-23.
Single, double, and quadruple replacement mutants showed V(max) comparable
to that of the wild type. On the basis of the values of internal and
external transport affinities (K(m)) for carnitine and of their comparison
with those measured in mitochondria, the recombinant CAC is oriented
unidirectionally in the liposomes, right side out compared to
mitochondria. Substitution of Cys-136 with Ser caused a nearly complete
loss of sensitivity of the CAC to N-ethylmaleimide, (2-aminoethyl)
methanethiosulfonate hydrobromide (MTSES), and other hydrophilic SH
reagents but not to the very hydrophobic N-phenylmaleimide. The wild-type
CAC and the mutants containing Cys-136 showed substrate protection against
NEM and MTSES inhibition and against NEM labeling. The data show that none
of the native cysteines is essential for the transport mechanism and that
Cys-136 is the major target of SH reagents and raise the hypothesis that
Cys-136 is accessible from the external medium and is located at, or near,
the substrate binding site. A model of the CAC is proposed in which the
matrix hydrophilic loop containing Cys-136 protrudes into the membrane
between the transmembrane domains of the protein. (literal)
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