Importing mitochondrial proteins: machineries and mechanisms

Abstract

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.

Figure 1. Principles of Mitochondrial Protein Biogenesis

(A) Cytosolic and mitochondrial protein synthesis. Most mitochondrial proteins are synthesized on cytosolic ribosomes and are imported into the organelle. About 1% of mitochondrial proteins are synthesized inside the organelle. (B) Sorting pathways of mitochondria. The translocase of the outer membrane (TOM complex) is the main entry gate into mitochondria. Subsequently, the precursor proteins follow different sorting pathways. MIA, mitochondrial intermembrane space assembly; OXA, insertase/export machinery of the inner membrane; SAM, sorting and assembly machinery; TIM22 complex, carrier translocase of the inner membrane; TIM23 complex, presequence translocase of the inner membrane. (Inset) The TOM complex consists of seven different subunits. The receptors Tom20, Tom22, and Tom70 recognize precursor proteins and transfer them to the central component, the channel-forming Tom40. Three small Tom proteins, Tom5, Tom6, and Tom7, are involved in the assembly and dynamics of the TOM complex. (Presequence-carrying precursor proteins, red; hydrophobic precursors with internal targeting signals, blue.)

Figure 2. Targeting and Sorting Signals of Mitochondrial Precursor Proteins

Mitochondrial precursor proteins contain cleavable or noncleavable targeting signals. The three main classes of signals are shown. The signals direct the precursor proteins to different sorting machineries. Presequences are usually cleaved off by the mitochondrial processing peptidase (MPP) in the matrix. Additional hydrophobic sorting signals can be removed by the inner membrane peptidase complex (IMP). The putative signal directing β-barrel precursors through the TOM complex is shown in light blue. The mitochondrial intermembrane space signal (MISS) for the MIA pathway is shown in red; MISS includes a cysteine residue of the precursor (shown here for the first cysteine). MIA, mitochondrial intermembrane space assembly; Mim1, putative insertase of the mitochondrial outer membrane; PAM, presequence translocase-associated motor; SAM, sorting and assembly machinery; Tim9–Tim10, chaperone complex in the intermembrane space; TIM22, carrier translocase of the inner membrane; TIM23, presequence translocase of the inner membrane; TOM, translocase of the outer membrane.

Figure 3. The Presequence Pathway to the Mitochondrial Inner Membrane and Matrix

(A) Forms of the presequence translocase of the inner membrane (TIM23 complex). (Left image) Motor-free TIM23 complex that can insert preproteins into the inner membrane. (Right image) TIM23 complex associated with PAM (presequence translocase-associated motor). The central chaperone that binds to preproteins is mtHsp70. The function of mtHsp70 is regulated by the cochaperones Pam16-Pam18, Tim44, Pam17, and the nucleotide exchange factor Mge1. The function of the individual components is described in Table 1. (B) Presequence-carrying preproteins are directed into the matrix by a sequential chain of binding sites. (C) Hypothetical model for the dynamic cooperation of the TIM23 complex of the inner membrane (IM) with the TOM complex of the outer membrane (OM), the respiratory chain complexes III (bc₁-complex) and IV (cytochrome c oxidase, COX), and the motor PAM. The membrane potential (Δψ) activates the Tim23 channel and exerts an electrophoretic effect on the positively charged presequences of preproteins. ATP drives the action of mtHsp70. The mitochondrial processing peptidase (MPP) removes the presequences.

Figure 4. Intermembrane Space Chaperones: The Carrier Pathway, and Machinery for Import and Assembly

(A) Carrier pathway to the inner mitochondrial membrane. The noncleavable precursors of hydrophobic metabolite carriers of the inner membrane are imported in several stages. Cytosolic chaperones guide the precursor (stage I) to the receptor Tom70 (stage II). The precursor is transported through the translocase of the outer membrane (TOM complex) in a loop formation and interacts with the Tim9–Tim10 chaperone complex (stage IIIa). Tim9–Tim10 guides the precursor through the intermembrane space (IMS) to the carrier translocase of the inner membrane (TIM22 complex with bound Tim9-10-12) (stage IIIb). The membrane potential (Δψ) promotes insertion of the precursor into the inner membrane via the TIM22 complex (stage IV), followed by assembly into the mature form of the carrier protein (stage V). (B) The machinery for import and assembly (MIA) of preproteins in the mitochondrial intermembrane space is required for small IMS proteins with cysteine motifs. The IMS receptor Mia40 binds to the precursor via a transient disulfide bond. The sulfhydryl oxidase Erv1 cooperates with Mia40 in the oxidation of the precursor protein. Erv1 reoxidizes Mia40 and transfers electrons to cytochrome c (Cyt. c). A third component, Hot13, assists in the oxidation of Mia40. (C) Comparison of the disulfide relay systems of the mitochondrial intermembrane space, the endoplasmic reticulum, and the bacterial periplasm. In each system, disulfide bonds are introduced into substrate proteins and electrons are removed (oxidation of substrate proteins). A disulfide generating enzyme oxidizes a disulfide carrier that in turn oxidizes the substrate.

Figure 5. Protein Insertion into the Mitochondrial Outer Membrane

(A) Sorting of β-barrel proteins. The precursors of β-barrel proteins are initially transported via the translocase of the outer membrane (TOM complex) into the intermembrane space (IMS). IMS chaperone complexes transfer the precursors to the sorting and assembly machinery (SAM complex). The two essential subunits, Sam50 and Sam35, cooperate in insertion of the precursors into the outer membrane. Steps 1–3 show the general pathway for sorting of β-barrel proteins. Steps 4 and 5 depict specific additional steps of the assembly pathway of the β-barrel protein, Tom40. Mim1 supports integration of the Tom40 precursor into the outer membrane; Tom40 then associates with Tom5 and a further Tom40 molecule, followed by Mdm10-supported assembly of the oligomeric TOM complex. (B) Insertion of α-helical proteins. Mitochondrial outer membrane proteins with α-helical transmembrane segments that typically function as sorting signals are imported by different pathways, depending on the location of the sorting signal. For different precursor proteins, a dependence on Mim1, Tom70, the TOM complex and/or SAM has been reported, but the mechanisms of membrane integration have not yet been defined. (C) Evolutionary conservation of β-barrel sorting from Gram-negative bacteria to mitochondria. Bacteria synthesize the precursor proteins in the cytoplasm. The precursors are translocated through the Sec machinery to the periplasm and guided by chaperones to the β-barrel assembly machinery (BAM) of the outer membrane. For mitochondrial β-barrel proteins, the site of synthesis and the initial translocation through the TOM complex are different from bacteria, yet the subsequent steps of chaperone-guided transfer through the IMS and insertion into the outer membrane by the SAM complex share characteristics with the bacterial system. Sam50 is homologous to the central BAM component BamA (Omp85/YaeT). However, differences are also apparent as the other SAM and BAM subunits, as well as the chaperones of the IMS and periplasm, are not homologous to each other, and the lipid composition of the mitochondrial outer membrane is different from that of the bacterial outer membrane.