The biochemical properties of the mitochondrial thiamine pyrophosphate carrier from Drosophila melanogaster.
The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides and cofactors across the inner mitochondrial membrane. The genome of Drosophila melanogaster encodes at least 46 members of this family. Only five of these have been characterized, whereas the transport functions of the remainder cannot be assessed with certainty. In the present study, we report the functional identification of two D. melanogaster genes distantly related to the human and yeast thiamine pyrophosphate carrier (TPC) genes as well as the corresponding expression pattern throughout development. Furthermore, the functional characterization of the D. melanogaster mitochondrial thiamine pyrophosphate carrier protein (DmTpc1p) is described. DmTpc1p was over-expressed in bacteria, the purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. Reconstituted DmTpc1p transports thiamine pyrophosphate and, to a lesser extent, pyrophosphate, ADP, ATP and other nucleotides. The expression of DmTpc1p in Saccharomyces cerevisiaeTPC1 null mutant abolishes the growth defect on fermentable carbon sources. The main role of DmTpc1p is to import thiamine pyrophosphate into mitochondria by exchange with intramitochondrial ATP and/or ADP.
Iacopetta
Domenico
D
Department of Pharmaco-Biology, University of Calabria, Arcavacata di Rende, Cosenza, Italy.
Carrisi
Chiara
C
De Filippis
Giuseppina
G
Calcagnile
Valeria M
VM
Cappello
Anna R
AR
Chimento
Adele
A
Curcio
Rosita
R
Santoro
Antonella
A
Vozza
Angelo
A
Dolce
Vincenza
V
Palmieri
Ferdinando
F
Capobianco
Loredana
L
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013835:Thiamine Pyrophosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D011994:Recombinant Proteins; D013835:Thiamine Pyrophosphate