Glutathione controls the redox state of the mitochondrial carnitine/acylcarnitine carrier Cys residues by glutathionylation.
BACKGROUND
The mitochondrial carnitine/acylcarnitine carrier (CAC) is essential for cell metabolism since it catalyzes the transport of acylcarnitines into mitochondria allowing the β-oxidation of fatty acids. CAC functional and structural properties have been characterized. Cys residues which could form disulfides suggest the involvement of CAC in redox switches.


METHODS
The effect of GSH and GSSG on the [(3)H]-carnitine/carnitine antiport catalyzed by the CAC in proteoliposomes has been studied. The Cys residues involved in the redox switch have been identified by site-directed mutagenesis. Glutathionylated CAC has been assessed by glutathionyl-protein specific antibody.


RESULTS
GSH led to increase of transport activity of the CAC extracted from liver mitochondria. A similar effect was observed on the recombinant CAC. The presence of glutaredoxin-1 (Grx1) accelerated the GSH activation of the recombinant CAC. The effect was more evident at 37°C. GSSG led to transport inhibition which was reversed by dithioerythritol (DTE). The effects of GSH and GSSG were studied on CAC Cys-mutants. CAC lacking C136 and C155 was insensitive to both reagents. Mutants containing these two Cys responded as the wild-type. Anti-glutathionyl antibody revealed the formation of glutathionylated CAC.


CONCLUSIONS
CAC is redox-sensitive and it is regulated by the GSH/GSSG couple. C136 and C155 are responsible for the regulation which occurs through glutathionylation.


CONCLUSIONS
CAC is sensitive to the redox state of the cell switching between oxidized and reduced forms in response to variation of GSSG and GSH concentrations.
CNR Institute of Biomembranes and Bioenergetics, via Amendola 165/A, 70126 Bari, Italy.
Nicola
N
Giangregorio
Ferdinando
F
Palmieri
Cesare
C
Indiveri
D000818:Animals; D001692:Biological Transport; D002331:Carnitine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D054477:Glutaredoxins / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000235:genetics / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000235:genetics / Q000378:metabolism; D008930:Mitochondria, Liver / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010084:Oxidation-Reduction; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism; D051381:Rats
D002352:Carrier Proteins; D054477:Glutaredoxins; D011510:Proteolipids; C116917:acylcarnitine; C020485:proteoliposomes; D005978:Glutathione; D003545:Cysteine; D002331:Carnitine; D019803:Glutathione Disulfide
1,4-piperazinediethanesulfonic acid; ANT; CAC; Carnitine/acylcarnitine carrier; DTE; GSH; GSSG; Glutathione; Glutathionylation; Mitochondrion; N-ethylmaleimide; NEM; NO; Pipes; SDS-PAGE; Site-directed mutagenesis; WT; adenine nucleotide transporter; carnitine/acylcarnitine carrier; dithioerythritol; glutathione; glutathione disulphide; nitric oxide; sodium dodecyl sulfate polyacrylamide gel electrophoresis; wild-type