Human mitochondrial C1-tetrahydrofolate synthase: submitochondrial localization of the full-length enzyme and characterization of a short isoform.
Mammalian mitochondrial C(1)-tetrahydrofolate (THF) synthase (MTHFDIL gene product) is a monofunctional 10-formyl-THF synthetase, lacking the 5,10-methylene-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities typically found in the trifunctional cytoplasmic proteins. Here, we report the submitochondrial localization of epitope-tagged human mitochondrial C(1)-THF synthase expressed in Chinese hamster ovary cells. Mitochondrial fractionation experiments show that human mitochondrial C(1)-THF synthase behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. Inner mitochondrial membrane association was also observed for the endogenous mitochondrial C(1)-THF synthase in adult rat spleen. We also purified and characterized the recombinant protein product (short isoform) of the alternatively spliced short transcript of the mitochondrial isozyme. Methylene-THF dehydrogenase assays confirmed that the short isoform is not enzymatically active. The purified short isoform was used in the production of polyclonal antibodies specific for the mitochondrial isozyme. These antibodies detected endogenous full-length mitochondrial C(1)-THF synthase in mitochondria from adult rat spleen and human placenta, confirming the expression of the mitochondrial isozyme in adult mammalian tissues.
Prasannan
Priya
P
Department of Chemistry and Biochemistry and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
Appling
Dean R
DR
D017398:Alternative Splicing; D000619:Aminohydrolases / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005574:Formate-Tetrahydrofolate Ligase / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000201:enzymology; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013154:Spleen / Q000201:enzymology
D007527:Isoenzymes; D024101:Mitochondrial Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D005574:Formate-Tetrahydrofolate Ligase