Dusanka Milenkovic

Importing Mitochondrial Proteins: Machineries and Mechanisms

Most mitochondrial proteins are synthesized on cytosolic ribosomes and must be imported across one or both mitochondrial membranes. There is an amazingly versatile set of machineries and mechanisms, and at least four different pathways, for the importing and sorting of mitochondrial precursor proteins. The translocases that catalyze these processes are highly dynamic machines driven by the membrane potential, ATP, or redox reactions, and they cooperate with molecular chaperones and assembly complexes to direct mitochondrial proteins to their correct destinations. Here, we discuss recent insights into the importing and sorting of mitochondrial proteins and their contributions to mitochondrial biogenesis.

LRPPRC is necessary for polyadenylation and coordination of translation of mitochondrial mRNAs

Regulation of mtDNA expression is critical for maintaining cellular energy homeostasis and may, in principle, occur at many different levels. The leucine‐rich pentatricopeptide repeat containing (LRPPRC) protein regulates mitochondrial mRNA stability and an amino‐acid substitution of this protein causes the French‐Canadian type of Leigh syndrome (LSFC), a neurodegenerative disorder characterized by complex IV deficiency. We have generated conditional Lrpprc knockout mice and show here that the gene is essential for embryonic development. Tissue‐specific disruption of Lrpprc in heart causes mitochondrial cardiomyopathy with drastic reduction in steady‐state levels of most mitochondrial mRNAs. LRPPRC forms an RNA‐dependent protein complex that is necessary for maintaining a pool of non‐translated mRNAs in mammalian mitochondria. Loss of LRPPRC does not only decrease mRNA stability, but also leads to loss of mRNA polyadenylation and the appearance of aberrant mitochondrial translation. The translation pattern without the presence of LRPPRC is misregulated with excessive translation of some transcripts and no translation of others. Our findings point to the existence of an elaborate machinery that regulates mammalian mtDNA expression at the post‐transcriptional level.

The morphology proteins Mdm12/Mmm1 function in the major β‐barrel assembly pathway of mitochondria

The β‐barrel proteins of mitochondria are synthesized on cytosolic ribosomes. The proteins are imported by the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been assumed that the SAMcore complex with the subunits Sam35, Sam37 and Sam50 represents the last import stage common to all β‐barrel proteins, followed by splitting in a Tom40‐specific route and a route for other β‐barrel proteins. We have identified new components of the β‐barrel assembly machinery and show that the major β‐barrel pathway extends beyond SAMcore. Mdm12/Mmm1 function after SAMcore yet before splitting of the major pathway. Mdm12/Mmm1 have been known for their role in maintenance of mitochondrial morphology but we reveal assembly of β‐barrel proteins as their primary function. Moreover, Mdm10, which functions in the Tom40‐specific route, can associate with SAMcore as well as Mdm12/Mmm1 to form distinct assembly complexes, indicating a dynamic exchange between the machineries governing mitochondrial β‐barrel assembly. We conclude that assembly of mitochondrial β‐barrel proteins represents a major function of the morphology proteins Mdm12/Mmm1.

Sam35 of the Mitochondrial Protein Sorting and Assembly Machinery Is a Peripheral Outer Membrane Protein Essential for Cell Viability

The mitochondrial outer membrane contains two integral proteins essential for cell viability, Tom40 of the translocase of the outer membrane (TOM complex) and Sam50 of the sorting and assembly machinery (SAM complex). Here we report the identification of Sam35, the first peripheral mitochondrial outer membrane protein that is essential for cell viability. Sam35 (encoded by the Saccharomyces cerevisiae ORF YHR083w) is a novel subunit of the SAM complex and is crucial for the assembly pathway of outer membrane β-barrel proteins, such as the precursors of Tom40 and porin. Sam35 is not required for the import of inner membrane or matrix targeted proteins. The presence of two essential proteins in the SAM complex, Sam35 and Sam50, indicates that it plays a central role in mitochondrial biogenesis.

Identification of the Signal Directing Tim9 and Tim10 into the Intermembrane Space of Mitochondria

The intermembrane space of mitochondria contains the specific mitochondrial intermembrane space assembly (MIA) machinery that operates in the biogenesis pathway of precursor proteins destined to this compartment. The Mia40 component of the MIA pathway functions as a receptor and binds incoming precursors, forming an essential early intermediate in the biogenesis of intermembrane space proteins. The elements that are crucial for the association of the intermembrane space precursors with Mia40 have not been determined. In this study, we found that a region within the Tim9 and Tim10 precursors, consisting of only nine amino acid residues, functions as a signal for the engagement of substrate proteins with the Mia40 receptor. Furthermore, the signal contains sufficient information to facilitate the transfer of proteins across the outer membrane to the intermembrane space. Thus, here we have identified the mitochondrial intermembrane space sorting signal required for delivery of proteins to the mitochondrial intermembrane space.

Biogenesis of the Mitochondrial TOM Complex Mim1 PROMOTES INSERTION AND ASSEMBLY OF SIGNAL-ANCHORED RECEPTORS

The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming β-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane α-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAMcore components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the β-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.

Mitochondrial Protein Sorting DIFFERENTIATION OF β-BARREL ASSEMBLY BY Tom7-MEDIATED SEGREGATION OF Mdm10

The mitochondrial outer membrane contains two distinct machineries for protein import and protein sorting that function in a sequential manner: the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex), which is dedicated to β-barrel proteins. The SAMcore complex consists of three subunits, Sam35, Sam37, and Sam50, that can associate with a fourth subunit, the morphology component Mdm10, to form the SAMholo complex. Whereas the SAMcore complex is required for the biogenesis of all β-barrel proteins, Mdm10 and the SAMholo complex play a selective role in β-barrel biogenesis by promoting assembly of Tom40 but not of porin. We report that Tom7, a conserved subunit of the TOM complex, functions in an antagonistic manner to Mdm10 in biogenesis of Tom40 and porin. We show that Tom7 promotes segregation of Mdm10 from the SAMholo complex into a low molecular mass form. Upon deletion of Tom7, the fraction of Mdm10 in the SAMholo complex is significantly increased, explaining the opposing functions of Tom7 and Mdm10 in β-barrel sorting. Thus the role of Tom7 is not limited to the TOM complex. Tom7 functions in mitochondrial protein biogenesis by a new mechanism, segregation of a sorting component, leading to a differentiation of β-barrel assembly.

Biogenesis of the essential Tim9-Tim10 chaperone complex of mitochondria: site-specific recognition of cysteine residues by the intermembrane space receptor Mia40.

The mitochondrial intermembrane space (IMS) contains an essential machinery for protein import and assembly (MIA). Biogenesis of IMS proteins involves a disulfide relay between precursor proteins, the cysteine-rich IMS protein Mia40 and the sulfhydryl oxidase Erv1. How precursor proteins are specifically directed to the IMS has remained unknown. Here we systematically analyzed the role of cysteine residues in the biogenesis of the essential IMS chaperone complex Tim9–Tim10. Although each of the four cysteines of Tim9, as well as of Tim10, is required for assembly of the chaperone complex, only the most amino-terminal cysteine residue of each precursor is critical for translocation across the outer membrane and interaction with Mia40. Mia40 selectively recognizes cysteine-containing IMS proteins in a site-specific manner in organello and in vitro. Our results indicate that Mia40 acts as a trans receptor in the biogenesis of mitochondrial IMS proteins.

Two Modular Forms of the Mitochondrial Sorting and Assembly Machinery Are Involved in Biogenesis of α-Helical Outer Membrane Proteins

The mitochondrial outer membrane contains two translocase machineries for precursor proteins--the translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The TOM complex functions as the main mitochondrial entry gate for nuclear-encoded proteins, whereas the SAM complex was identified according to its function in the biogenesis of beta-barrel proteins of the outer membrane. The SAM complex is required for the assembly of precursors of the TOM complex, including not only the beta-barrel protein Tom40 but also a subset of alpha-helical subunits. While the interaction of beta-barrel proteins with the SAM complex has been studied in detail, little is known about the interaction between the SAM complex and alpha-helical precursor proteins. We report that the SAM is not static but that the SAM core complex can associate with different partner proteins to form two large SAM complexes with different functions in the biogenesis of alpha-helical Tom proteins. We found that a subcomplex of TOM, Tom5-Tom40, associates with the SAM core complex to form a new large SAM complex. This SAM-Tom5/Tom40 complex binds the alpha-helical precursor of Tom6 after the precursor has been inserted into the outer membrane in an Mim1 (mitochondrial import protein 1)-dependent manner. The second large SAM complex, SAM-Mdm10 (mitochondrial distribution and morphology protein), binds the alpha-helical precursor of Tom22 and promotes its membrane integration. We suggest that the modular composition of the SAM complex provides a flexible platform to integrate the sorting pathways of different precursor proteins and to promote their assembly into oligomeric complexes.

TWINKLE is an essential mitochondrial helicase required for synthesis of nascent D-loop strands and complete mtDNA replication

Replication of the mammalian mitochondrial DNA (mtDNA) is dependent on the minimal replisome, consisting of the heterotrimeric mtDNA polymerase (POLG), the hexameric DNA helicase TWINKLE and the tetrameric single-stranded DNA-binding protein (mtSSB). TWINKLE has been shown to unwind DNA during the replication process and many disease-causing mutations have been mapped to its gene. Patients carrying Twinkle mutations develop multiple deletions of mtDNA, deficient respiratory chain function and neuromuscular symptoms. Despite its importance in human disease, it has been unclear whether TWINKLE is the only replicative DNA helicase in mammalian mitochondria. Furthermore, a substantial portion of mtDNA replication events is prematurely terminated at the end of mitochondrial control region (D-loop) and it is unknown whether TWINKLE also has a role in this abortive replication. Here, we present a conditional mouse knockout for Twinkle and demonstrate that TWINKLE is essential for mouse embryonic development and thus is the only replicative DNA helicase in mammalian mitochondria. Conditional knockout of Twinkle results in severe and rapid mtDNA depletion in heart and skeletal muscle. No replication intermediates or deleted mtDNA molecules are observed after Twinkle knockout, suggesting that TWINKLE once loaded is very processive. We also demonstrate that TWINKLE is essential for nascent H-strand synthesis in the D-loop, thus showing that there is no separate DNA helicase responsible for replication of this region. Our data thus suggest that the relative levels of abortive D-loop synthesis versus complete mtDNA replication are regulated and may provide a mechanism to control progression to complete mtDNA replication.