Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and sorting include the TIM23 translocase responsible for the transfer of precursor proteins into the matrix, and the mitochondrial intermembrane space import and assembly (MIA) machinery required for the biogenesis of intermembrane space proteins. Dysfunction of mitochondrial protein sorting pathways results in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death. The cellular responses caused by accumulation of mitochondrial precursor proteins in the cytosol are mainly unknown. Here we present a comprehensive picture of the changes in the cellular transcriptome and proteome in response to a mitochondrial import defect and precursor over-accumulation stress. Pathways were identified that protect the cell against mitochondrial biogenesis defects by inhibiting protein synthesis and by activation of the proteasome, a major machine for cellular protein clearance. Proteasomal activity is modulated in proportion to the quantity of mislocalized mitochondrial precursor proteins in the cytosol. We propose that this type of unfolded protein response activated by mistargeting of proteins (UPRam) is beneficial for the cells. UPRam provides a means for buffering the consequences of physiological slowdown in mitochondrial protein import and for counteracting pathologies that are caused or contributed by mitochondrial dysfunction.

Mitochondrial precursor proteins are synthesized in the cytosol and subsequently imported into mitochondria. The import of mitochondrial intermembrane space proteins is coupled with their oxidative folding and governed by the mitochondrial intermembrane space import and assembly (MIA) pathway. The cytosolic steps that precede mitochondrial import are not well understood. We identified a role for the ubiquitin-proteasome system in the biogenesis of intermembrane space proteins. Interestingly, the function of the ubiquitin-proteasome system is not restricted to conditions of mitochondrial protein import failure. The ubiquitin-proteasome system persistently removes a fraction of intermembrane space proteins under physiological conditions, acting as a negative regulator in the biogenesis of this class of proteins. Thus, the ubiquitin-proteasome system plays an important role in determining the levels of proteins targeted to the intermembrane space of mitochondria.A s many as 800 to 1,000 proteins in a simple eukaryote, Saccharomyces cerevisiae, are required for mitochondria to perform their essential metabolic and regulatory functions in the cell (1-3). The majority of mitochondrial proteins are synthesized on cytosolic ribosomes and targeted to mitochondria. This process involves the main entry gate, formed by the translocase of the outer mitochondrial membrane (TOM) complex. After passing the TOM complex, mitochondrial precursor proteins are directed to their final destinations by specialized translocation machineries and pathways that drive the import of precursor proteins into the mitochondrial matrix, inner mitochondrial membrane, outer mitochondrial membrane, and intermembrane space (IMS) of mitochondria (4-7). The proteins that reside in the IMS are critical for metabolic functions (e.g., energy conversion), mitochondrial biogenesis (e.g., the transport of lipids and proteins and assembly of protein membrane complexes), and regulatory processes at the cellular level (e.g., programmed cell death) (8)(9)(10)(11)(12). A large fraction of mitochondrial IMS proteins are imported via the foldingtrap mechanism, executed by the mitochondrial intermembrane space import and assembly (MIA) pathway (13-16).Intermembrane space proteins that follow the MIA pathway are small cysteine-rich proteins that comprise two major families with characteristic twin cysteine motifs: CX 3 C (small Tim proteins) (17) and CX 9 C (12, 18, 19). The mechanism utilized by the MIA pathway is unique among mitochondrial transport pathways but also in the context of cellular protein transport machineries (13-16). The most distinctive feature of the MIA pathway is the transfer of disulfide bonds into the incoming precursors, leading to their oxidative folding and to trapping of mature proteins in the IMS (20-24). The specificity of disulfide transfer and oxidative folding is maintained by the recognition by Mia40 of the mitochondrial IMS sorting signal, called MISS/ITS, in the precursor proteins (25, 26). Mia40 serves not only as a receptor on the...

The MIA pathway governs the localization and oxidative folding of intermembrane space proteins. This study reports that the MIA pathway is involved in the transport of mitochondrial inner membrane protein Tim22, thereby broadening the known functions of MIA to the biogenesis of inner membrane proteins.

a b s t r a c tMitochondria are involved in many essential cellular activities. These broad functions explicate the need for the well-orchestrated biogenesis of mitochondrial proteins to avoid death and pathological consequences, both in unicellular and more complex organisms. Yeast as a model organism has been pivotal in identifying components and mechanisms that drive the transport and sorting of nuclear-encoded mitochondrial proteins. The machinery components that are involved in the import of mitochondrial proteins are generally evolutionarily conserved within the eukaryotic kingdom. However, topological and functional differences have been observed. We review the similarities and differences in mitochondrial translocases from yeast to human. Additionally, we provide a systematic overview of the contribution of mitochondrial import machineries to human pathologies, including cancer, mitochondrial diseases, and neurodegeneration.

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