Title: Cellular maturation of mitochondrial molybdoenzymes
Abstract: The molybdenum cofactor (Moco) is an essential component present in nearly all domains of life. In mammals, Moco is part of four currently known enzymes and constitutes a crucial redox-active center involved in a number of fundamental cellular reactions. Moco-dependent enzymes are present in the cytosol but also in or at mitochondria, where Moco is integrated into sulfite oxidase (SO) and the mitochondrial amidoxime-reducing component (mARC), respectively. The family of mitochondrial Moco-enzymes is of particular interest considering the cytosolic synthesis of enzymes and cofactor, which requires a coordinated mitochondrial transport and assembly process. In the current study, the mitochondrial maturations of SO and mARC1 were thus analyzed to obtain a mechanistic understanding of the processes starting with the cytosolic syntheses of apo-proteins all the way to the formation of the mature mitochondrial enzymes.
The first part of this work uncovered the cellular assembly of SO, a soluble protein of the mitochondrial intermembrane space, and revealed a Moco-dependent mitochondrial targeting mechanism. In spite of its functional bipartite N-terminal targeting signal, about 70% of SO mislocalized to the cytosol if Moco was not present. Following the identification of SO processing by the inner membrane peptidase (IMP) complex, prevention of this cleavage and thus anchoring of SO in the inner mitochondrial membrane resulted in an efficient mitochondrial targeting even in absence of Moco. SO was thereby identified to undergo a reverse translocation to the cytosol in absence of Moco, which is required to trap SO in the intermembrane space and to constitute in addition a vectorial driving force for completion of SO translocation across the TOM complex. The integration of Moco is not only essential for correct sub-mitochondrial localization, but also a prerequisite for in vivo heme integration and homodimerization of SO. In conclusion, the identified molecular hierarchy of SO maturation represents a novel link between the canonical pre-sequence pathway and folding-trap mechanisms of mitochondrial import.
The other mitochondrial Moco-enzyme mARC1 was recently discovered and its sub-mitochondrial localization had remained unclear. In the second part of this study, mARC1 was shown to be localized to the outer mitochondrial membrane. As a result of the translocation process, the C-terminal catalytic core of the protein remains exposed to the cytosol and confers an N(in)-C(out) membrane orientation of mARC1. This localization is mediated by the N-terminal domain of the enzyme, being composed of a classical but weak N-terminal targeting signal and a downstream transmembrane domain. Thereby, the transmembrane domain of mARC1 is sufficient for mitochondrial targeting, while the N-terminal targeting signal seems to function as a supportive receptor for the outer mitochondrial membrane. According to its localization and targeting mechanism, mARC1 is classified as a novel signal-anchored protein. Considering the membrane integration of mARC1, an SO-similar demand of Moco for mitochondrial retention of mARC1 is not required and its N-terminal targeting motifs are sufficient for adequate mitochondrial localization. During mitochondrial import, mARC1 is not processed and membrane integration proceeds membrane potential independently but requires external ATP, which finally results in the assembly of mARC1 into high-oligomeric protein complexes.
Publication Year: 2012
Publication Date: 2012-01-01
Language: en
Type: dissertation
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