The pyrimidine nucleotide carrier PNC1 and mitochondrial trafficking of thymidine phosphates in cultured human cells.
In cycling cells cytosolic de novo synthesis of deoxynucleotides is the main source of precursors for mitochondrial (mt) DNA synthesis. The transfer of deoxynucleotides across the inner mt membrane requires protein carriers. PNC1, a SLC25 family member, exchanges pyrimidine nucleoside triphosphates in liposomes and its downregulation decreases mtUTP concentration in cultured cells. By an isotope-flow protocol we confirmed transport of uridine nucleotides by PNC1 in intact cultured cells and investigated PNC1 involvement in the mt trafficking of thymidine phosphates. Key features of our approach were the manipulation of PNC1 expression by RNA interference or inducible overexpression, the employment of cells proficient or deficient for cytosolic thymidine kinase (TK1) to distinguish the direction of flow of thymidine nucleotides across the mt membrane during short pulses with [(3)H]-thymidine, the determination of mtdTTP specific radioactivity to quantitate the rate of mtdTTP export to the cytoplasm. Downregulation of PNC1 in TK1(-) cells increased labeled dTTP in mitochondria due to a reduced rate of export. Overexpression of PNC1 in TK1(+) cells increased mtdTTP pool size and radioactivity, suggesting an involvement in the import of thymidine phosphates. Thus PNC1 is a component of the network regulating the mtdTTP pool in human cells.
Department of Biology, University of Padova, Via Ugo Bassi 58B, I-35131 Padova, Italy.
Elisa
E
Franzolin
Cristina
C
Miazzi
Miriam
M
Frangini
Elisa
E
Palumbo
Chiara
C
Rampazzo
Vera
V
Bianchi
D001692:Biological Transport; D015153:Blotting, Western; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D033704:Nucleotide Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013937:Thymidine Kinase / Q000502:physiology*; D013942:Thymine Nucleotides / Q000378:metabolism*
D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D033704:Nucleotide Transport Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C483480:SLC25A33 protein, human; D013942:Thymine Nucleotides; D013937:Thymidine Kinase; C092717:thymidine kinase 1; C024157:thymidine 5'-triphosphate