Abstract
Heme is an essential cofactor for most organisms and all metazoans. While the individual enzymes involved in synthesis and utilization of heme are fairly well known, less is known about the intracellular trafficking of porphyrins and heme, or regulation of heme biosynthesis via protein complexes. To better understand this process we have undertaken a study of macromolecular assemblies associated with heme synthesis. Herein we have utilized mass spectrometry with coimmunoprecipitation of tagged enzymes of the heme biosynthetic pathway in a developing erythroid cell culture model to identify putative protein partners. The validity of these data obtained in the tagged protein system is confirmed by normal porphyrin/heme production by the engineered cells. Data obtained are consistent with the presence of a mitochondrial heme metabolism complex which minimally consists of ferrochelatase, protoporphyrinogen oxidase and aminolevulinic acid synthase-2. Additional proteins involved in iron and intermediary metabolism as well as mitochondrial transporters were identified as potential partners in this complex. The data are consistent with the known location of protein components and support a model of transient protein-protein interactions within a dynamic protein complex.
Highlights
Metabolons are intracellular complexes of enzymes that facilitate channeling of substrates within a biochemical pathway[1]
We report that the mitochondrially-located heme synthesis enzymes Alas2, protoporphyrinogen oxidase (Ppox) and Fech interact with each other as well as with additional protein partners, and appear to exist in a complex in the mitochondrion
The amino terminal FLAG-PBGS, -HMBS, -UROS and-UROD did not result in the recovery of any other cytosolic heme biosynthetic enzyme suggesting that if a cytosolic heme biosynthetic metabolon forms, it is not via Murine protein pbgs hmbs uros urod
Summary
Metabolons are intracellular complexes of enzymes that facilitate channeling of substrates within a biochemical pathway[1]. These protein complexes serve multiple roles including the reduction or prevention of competition for metabolites by intersecting pathways (metabolic interference), increasing flux while keeping the overall concentration of pathway reactants low, and protecting cells from damage attributable to reactive metabolic intermediates[2]. Mitochondrial Heme Metabolism Complex analysis, decision to publish, or preparation of the manuscript
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