title: The Mtm1p carrier and pyridoxal 5'-phosphate cofactor trafficking in yeast mitochondria.
abstract: Biochemical communication between the cytoplasmic and mitochondrial subsystems of the cell depends on solute carriers in the mitochondrial inner membrane that transport metabolites between the two compartments. We have expressed and purified a yeast mitochondrial carrier protein (Mtm1p, YGR257cp), originally identified as a manganese ion carrier, for biochemical characterization aimed at resolving its function. High affinity, stoichiometric pyridoxal 5'-phosphate (PLP) cofactor binding was characterized by fluorescence titration and calorimetry, and the biochemical effects of mtm1 gene deletion on yeast mitochondria were investigated. The PLP status of the mitochondrial proteome (the mitochondrial 'PLP-ome') was probed by immunoblot analysis of mitochondria isolated from wild type (MTM1(+)) and knockout (MTM1(-)) yeast, revealing depletion of mitochondrial PLP in the latter. A direct activity assay of the enzyme catalyzing the first committed step of heme biosynthesis, the PLP-dependent mitochondrial enzyme 5-aminolevulinate synthase, extends these results, providing a specific example of PLP cofactor limitation. Together, these experiments support a role for Mtm1p in mitochondrial PLP trafficking and highlight the link between PLP cofactor transport and iron metabolism, a remarkable illustration of metabolic integration.
authors: Institute of Environmental Health, Division of Environmental and Biomolecular Systems, Oregon Health and Science University, Portland, OR 97239-3098, USA.
authors: Mei M
authors: MM
authors: Whittaker
authors: Vollum Institute, Oregon Health and Science University, Portland, OR 97239-3098, USA.
authors: Aravind
authors: A
authors: Penmatsa
authors: Institute of Environmental Health, Division of Environmental and Biomolecular Systems, Oregon Health and Science University, Portland, OR 97239-3098, USA. Electronic address: whittakj@ohsu.edu.
authors: James W
authors: JW
authors: Whittaker
mesh_terms: D000624:5-Aminolevulinate Synthetase / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D007501:Iron / Q000378:metabolism; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D024101:Mitochondrial Proteins; C000597154:Mtm1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D011732:Pyridoxal Phosphate; D007501:Iron; D000624:5-Aminolevulinate Synthetase
keywords: Heme biosynthesis; Iron homeostasis; Mitochondrial carrier protein; Pyridoxal 5′-phosphate
references: J Biol Chem. 1978 Mar 25;253(6):2026-32
references: 632253
references: Metallomics. 2013 Jun;5(6):656-72
references: 23598994
references: Methods Cell Biol. 2007;80:45-64
references: 17445688
references: Arch Biochem Biophys. 1991 Jul;288(1):79-86
references: 1898027
references: Biochim Biophys Acta. 2008 Jul-Aug;1777(7-8):564-78
references: 18406340
references: Cell Metab. 2009 Aug;10(2):119-30
references: 19656490
references: J Biol Chem. 1981 May 10;256(9):4478-88
references: 7217092
references: Proc Natl Acad Sci U S A. 2003 Sep 2;100(18):10353-7
references: 12890866
references: J Biol Chem. 2005 Jun 17;280(24):22715-20
references: 15851472
references: J Biol Chem. 2014 Jan 17;289(3):1377-87
references: 24257746
references: Photochem Photobiol. 2010 Jan-Feb;86(1):39-46
references: 19930123
references: Protein Expr Purif. 2005 May;41(1):207-34
references: 15915565
references: Arch Biochem Biophys. 2012 Jul 15;523(2):191-7
references: 22561997
references: Int J Pept Protein Res. 1975;7(5):411-6
references: 1184290
references: Protein Expr Purif. 2014 Jan;93:77-86
references: 24184947
references: Biochemistry. 1999 Mar 2;38(9):2636-46
references: 10052933
references: Curr Biol. 2006 Jul 25;16(14):R551-60
references: 16860735
references: Methods Enzymol. 1979;62:528-51
references: 374981
references: EMBO J. 2006 Apr 19;25(8):1775-83
references: 16601688
references: Nucleic Acids Res. 2001 Feb 15;29(4):914-20
references: 11160923
references: J Biol Chem. 2007 Oct 12;282(41):29927-35
references: 17702748
references: EMBO Rep. 2003 Sep;4(9):850-4
references: 12949584
references: Nature. 2003 Nov 6;426(6962):39-44
references: 14603310
references: Methods Mol Biol. 2008;457:141-9
references: 19066024
references: J Biol Chem. 1956 Mar;219(1):435-46
references: 13295297
references: J Bioenerg Biomembr. 2000 Feb;32(1):67-77
references: 11768764
references: J Immunol Methods. 1985 Apr 8;78(1):87-94
references: 2580028
references: Science. 1999 Aug 6;285(5429):901-6
references: 10436161
references: PLoS One. 2007;2(10):e1078
references: 17957260
references: BMC Bioinformatics. 2009;10:273
references: 19723314
references: FEBS J. 2010 May;277(9):2157-70
references: 20370823
references: J Vis Exp. 2009;(30). pii: 1417. doi: 10.3791/1417
references: 19704406
references: J Biol Chem. 1982 Jul 25;257(14):8163-70
references: 7085661
references: Annu Rev Biochem. 2007;76:673-8
references: 17313357
references: Adv Microb Physiol. 2005;50:41-101
references: 16221578
references: Eur J Biochem. 1984 Aug 1;142(3):551-7
references: 6381051
references: Methods Enzymol. 1995;260:349-69
references: 8592459
references: Essays Biochem. 2010;47:37-52
references: 20533899
references: J Biol Chem. 2004 Aug 27;279(35):36906-15
references: 15220327
references: Mol Aspects Med. 2013 Apr-Jun;34(2-3):465-84
references: 23266187
references: J Biol Chem. 2014 Mar 28;289(13):8735-41
references: 24515117
references: J Biol Chem. 2001 Jun 8;276(23):19855-61
references: 11278996
references: Biochim Biophys Acta. 2006 Jul;1763(7):723-36
references: 16839620
references: Curr Protoc Mol Biol. 2008 Apr;Chapter 13:Unit 13.2
references: 18425759
references: Methods Enzymol. 1997;281:309-16
references: 9250995
references: IUBMB Life. 2009 Jan;61(1):40-6
references: 18816452