Mitochondrial proteins: from biogenesis to functional networks

Abstract

Mitochondria are essential for the viability of eukaryotic cells as they perform crucial functions in bioenergetics, metabolism and signalling and have been associated with numerous diseases. Recent functional and proteomic studies have revealed the remarkable complexity of mitochondrial protein organization. Protein machineries with diverse functions such as protein translocation, respiration, metabolite transport, protein quality control and the control of membrane architecture interact with each other in dynamic networks. In this Review, we discuss the emerging role of the mitochondrial protein import machinery as a key organizer of these mitochondrial protein networks. The preprotein translocases that reside on the mitochondrial membranes not only function during organelle biogenesis to deliver newly synthesized proteins to their final mitochondrial destination but also cooperate with numerous other mitochondrial protein complexes that perform a wide range of functions. Moreover, these protein networks form membrane contact sites, for example, with the endoplasmic reticulum, that are key for integration of mitochondria with cellular function, and defects in protein import can lead to diseases.

Figure 1. Overview of mitochondria and their functions.

Mitochondria consist of four compartments: outer membrane, intermembrane space, inner membrane and matrix. A large variety of functions have been assigned to mitochondrial proteins and protein complexes and are indicated in the figure: energy metabolism with respiration and synthesis of ATP; metabolism of amino acids, lipids and nucleotides; biosynthesis of iron-sulfur (Fe/S) clusters and cofactors; expression of the mitochondrial genome; quality control and degradation processes including mitophagy and apoptosis; signaling and redox processes; membrane architecture and dynamics; and the import and processing of precursor proteins that are synthesized on cytosolic ribosomes. AAA, ATP-dependent proteases of the inner membrane; E3, ubiquitin ligase; Msp1, mitochondrial sorting of proteins, extracts mistargeted proteins; TCA, tricarboxylic acid cycle; Ub, ubiquitin.

Figure 2. Protein import pathways into mitochondria.

Five major pathways of mitochondrial protein import have been identified. The protein import machineries have been well conserved from fungi (shown in this figure) to mammals (shown in BOX 4). First, the presequence pathway transports presequence-carrying cleavable preproteins through the translocase of the outer membrane (TOM) and the presequence translocase of the inner membrane (TIM23) with the presequence translocase-associated motor (PAM). The membrane potential Δψ across the inner membrane (IM) activates the TIM23 channel and drives translocation of the positively charged presequences into the matrix. The presequences are removed by the mitochondrial processing peptidase (MPP) and additional proteolytic processing can occur by the intermediate cleaving peptidase (Icp55) or the octapeptidyl peptidase (Oct1). Cleavable IM proteins are either laterally released from the TIM23 complex or are transported via the matrix and inserted into the IM by the oxidase assembly (Oxa1) insertase. IM proteins synthesized on mitochondrial ribosomes are also inserted by Oxa1. Second, cysteine-rich proteins destined for the intermembrane space (IMS) are imported through the TOM complex and are recognized by the mitochondrial IMS import and assembly protein (Mia40) that functions as oxidoreductase to insert disulfide bonds into the imported proteins. The sulfhydryl oxidase Erv1 forms a disulfide relay with Mia40, transferring disulfides from Erv1 to Mia40 to imported proteins. Third, the precursors of non-cleavable IM proteins such as the carrier proteins are imported by the TOM complex, followed by transfer to the small TIM chaperones in the IMS and insertion into the IM by the TIM22 carrier translocase. Fourth, the precursors of outer membrane (OM) β-barrel proteins use the TOM complex and small TIM chaperones and are inserted into the OM by the sorting and assembly machinery (SAM). Fifth, many OM proteins with α-helical transmembrane segments are inserted into the membrane by the mitochondrial import (MIM) complex. α-helical OM proteins typically do not use the Tom40 channel, but Tom70 can be involved in their recognition. Inset, assessment of absolute copy numbers of mitochondrial proteins in a respiring yeast cell. The porin isoform Por1 of the OM is one of the most abundant mitochondrial proteins, whereas the isoform Por2 is one of the least abundant proteins.

Figure 3. Interaction network of respiratory complexes, biogenesis and quality control machineries.

Supercomplexes of the mitochondrial respiratory chain are integrated into functional networks with the presequence translocase TIM23 (see BOX 2) and the AAA proteases of the inner membrane (IM). The ATP-dependent AAA proteases not only degrade several IM proteins, but also selected proteins of the matrix, intermembrane space (IMS) and outer membrane (OM), functioning as a quality control system of mitochondria. Several respiratory chain-AAA linker proteins, AAA-substrate adapter proteins, and assembly factors for respiratory supercomplexes were identified in fungi (indicated by dashed lines). The coenzyme Q (CoQ, Q) biosynthetic complex on the matrix side of the IM provides CoQ for the respiratory chain and further enzymes. The precursors of the CoQ complex are imported by the TOM and TIM machineries. Proteolytic processing in the matrix can involve two steps like for the precursor of Coq5. The mitochondrial respiratory chain is a main source for the generation of ROS that can exert harmful effects but also function in signaling. Targeting of the cytosolic translation machinery by ROS leads to a decreased protein synthesis, providing a link between the status of the respiratory chain and protein biogenesis.

Figure 4. Mitochondrial organizing network.

The mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane (IM) and the protein translocases TOM and SAM of the outer membrane (OM) form the core of a large ER-mitochondria organizing network (ERMIONE) that includes multiple dynamic interactions: to the ER-mitochondria encounter structure (ERMES); to further ER-mitochondria contact sites that involve the receptor Tom70 and IP3 receptors or the lipid transfer protein Lam6/Ltc1, as well as to vacuole-mitochondria contact sites (including Tom40 and the bridging protein Vps39/Vam6); to the kinase PINK1 and the metabolite channel VDAC (porin); to the mitochondrial intermembrane space (IMS) protein import and assembly system (Mia40); to respiratory chain complexes, the F₁F_O-ATP synthase, and the fusion protein OPA1 of the IM; and to mtDNA nucleoids (with the mtDNA packaging factor, termed mitochondrial transcription factor A, TFAM) of the matrix. Most components shown have been functionally conserved from yeast to humans; proteins that have been characterized in fungi only are indicated by a dashed border, whereas proteins that have been characterized in metazoans so far are bordered in red. In sum, ERMIONE forms a membrane-spanning system for the coordination of protein and lipid biogenesis, energetics, inheritance and quality control of mitochondria.