Nikolaus Pfanner

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.

The proteome of Saccharomyces cerevisiae mitochondria

We performed a comprehensive approach to determine the proteome of Saccharomyces cerevisiae mitochondria. The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS. From >20 million MS spectra, 750 different proteins were identified, indicating an involvement of mitochondria in numerous cellular processes. All known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and the stable mitochondria-encoded proteins were found. Based on the mitochondrial proteins described in the literature so far, we calculate that the identified proteins represent ≈90% of all mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The mitochondrial proteome will provide an important database for the analysis of new mitochondrial and mitochondria-associated functions and the characterization of mitochondrial diseases.

Versatility of the mitochondrial protein import machinery

The vast majority of mitochondrial proteins are synthesized in the cytosol and are imported into mitochondria by protein machineries located in the mitochondrial membranes. It has become clear that hydrophilic as well as hydrophobic preproteins use a common translocase in the outer mitochondrial membrane, but diverge to two distinct translocases in the inner membrane. The translocases are dynamic, high-molecular-weight complexes that have to provide specific means for the recognition of preproteins, channel formation and generation of import-driving forces.

Mitochondrial protein import: from proteomics to functional mechanisms

Mitochondria contain ∼1,000 different proteins, most of which are imported from the cytosol. Two import pathways that direct proteins into the mitochondrial inner membrane and matrix have been known for many years. The identification of numerous new transport components in recent proteomic studies has led to novel mechanistic insight into these pathways and the discovery of new import pathways into the outer membrane and intermembrane space. Protein translocases do not function as independent units but are integrated into dynamic networks and are connected to machineries that function in bioenergetics, mitochondrial morphology and coupling to the endoplasmic reticulum.

Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins

The mitochondrial outer membrane contains machinery for the import of preproteins encoded by nuclear genes. Eight different Tom (translocase of outer membrane) proteins have been identified that function as receptors and/or are related to a hypothetical general import pore. Many mitochondrial membrane channel activities have been described, including one related to Tim23 of the inner-membrane protein-import system; however, the pore-forming subunit(s) of the Tom machinery have not been identified until now. Here we describe the expression and functional reconstitution of Tom40, an integral membrane protein with mainly β-sheet structure. Tom40 forms a cation-selective high-conductance channel that specifically binds to and transports mitochondrial-targeting sequences added to the cis side of the membrane. We conclude that Tom40 is the pore-forming subunit of the mitochondrial general import pore and that it constitutes a hydrophilic, ∼22 Å wide channel for the import of preproteins.

Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins

Mitochondria import nuclear‐encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.

Machinery for protein sorting and assembly in the mitochondrial outer membrane

Mitochondria contain translocases for the transport of precursor proteins across their outer and inner membranes. It has been assumed that the translocases also mediate the sorting of proteins to their submitochondrial destination. Here we show that the mitochondrial outer membrane contains a separate sorting and assembly machinery (SAM) that operates after the translocase of the outer membrane (TOM). Mas37 forms a constituent of the SAM complex. The central role of the SAM complex in the sorting and assembly pathway of outer membrane proteins explains the various pleiotropic functions that have been ascribed to Mas37 (refs 4, 11–15). These results suggest that the TOM complex, which can transport all kinds of mitochondrial precursor proteins, is not sufficient for the correct integration of outer membrane proteins with a complicated topology, and instead transfers precursor proteins to the SAM complex.

Mitochondrial import and the twin-pore translocase

The mitochondrial inner membrane is rich in multispanning integral membrane proteins, most of which mediate the vital transport of molecules between the matrix and the intermembrane space. The correct transport and membrane insertion of such proteins is essential for maintaining the correct exchange of molecules between mitochondria and the rest of the cell. Mitochondria contain several specific complexes — known as translocases — that translocate precursor proteins. Recent analysis of the inner-membrane, twin-pore protein translocase (TIM22 complex) allows a glimpse of the molecular mechanisms by which this machinery triggers protein insertion using the membrane potential as an external driving force.

Global Analysis of the Mitochondrial N-Proteome Identifies a Processing Peptidase Critical for Protein Stability

Many mitochondrial proteins are synthesized with N-terminal presequences that are removed by specific peptidases. The N-termini of the mature proteins and thus peptidase cleavage sites have only been determined for a small fraction of mitochondrial proteins and yielded a controversial situation for the cleavage site specificity of the major mitochondrial processing peptidase (MPP). We report a global analysis of the N-proteome of yeast mitochondria, revealing the N-termini of 615 different proteins. Significantly more proteins than predicted contained cleavable presequences. We identified the intermediate cleaving peptidase Icp55, which removes an amino acid from a characteristic set of MPP-generated N-termini, solving the controversial situation of MPP specificity and suggesting that Icp55 converts instable intermediates into stable proteins. Our results suggest that Icp55 is critical for stabilization of the mitochondrial proteome and illustrate how the N-proteome can serve as rich source for a systematic analysis of mitochondrial protein targeting, cleavage and turnover.

Mitochondrial Presequence Translocase: Switching between TOM Tethering and Motor Recruitment Involves Tim21 and Tim17

The presequence translocase of the inner mitochondrial membrane (TIM23 complex) operates at a central junction of protein import. It accepts preproteins from the outer membrane TOM complex and directs them to inner membrane insertion or, in cooperation with the presequence translocase-associated motor (PAM), to the matrix. Little is known of how the TIM23 complex coordinates these tasks. We have identified Tim21 (YGR033c) that interacts with the TOM complex. Tim21 is specific for a TIM23 form that cooperates with TOM and promotes inner membrane insertion. Protein translocation into the matrix requires a switch to a Tim21-free, PAM bound presequence translocase. Tim17 is crucial for the switch by performing two separable functions: promotion of inner membrane insertion and binding of Pam18 to form the functional TIM-PAM complex. Thus, the presequence translocase is not a static complex but switches between TOM tethering and PAM binding in a reaction cycle involving Tim21 and Tim17.