y between cells and even within a cell, depending on the metabolic states. Cardiomyocytes and neurons, due to their high energy demands, have greater amounts. Mitochondria are dynamic and form reticular networks to assure efficient distribution of mitochondrial DNA (mtDNA) and proteins. The fission and fusion of mitochondria are features of mitochondria dynamics along with their trafficking along microtubule routes in higher organisms (actin in yeast). Mitochondria dynamics and the mitochondrial autophagy (mitophagy) pathways are intimately connected. Fission is believed to be a pre-requisite for mitophagy, allowing for the delineation of individual, damaged mitochondria while mitophagy targets components of the fusion apparatus and of transport for degradation. Of the ~1,500 mitochondrial proteins in humans (~1,000 in yeast), only 13 (8 in yeast) are encoded in the mtDNA, the rest (~99%), have to be imported. As such, the mitochondrial protein import pathway impacts on every aspect of mitochondrial function, including mitochondria dynamics and autophagy.

Currently, five pathways of mitochondrial protein import deliver the nuclear-encoded protein precursors to the appropriate mitochondrial sub-compartments using several translocases/systems. The translocase of the outer mitochondrial membrane (TOM) is a major gate for protein import and is shared by almost all pathways. Beyond TOM, they follow distinct routes that inter-cooperate. Proteins with a cleavable presequence peptide are targeted to the mitochondrial matrix or embedded in the inner mitochondrial membrane (IMM) via TIM23 translocase, further assisted by the PAM motor system in the case of matrix proteins. Many metabolite carrier proteins are synthesized without a presequence and they are imported into IMM using TIM22 translocase. OXA system inserts the mitochondria-encoded proteins into the IMM. Cysteine-rich proteins destined for the mitochondrial intermembrane space (IMS) are imported via the MIA system. The outer mitochondrial membrane (OMM) proteins are of two types: the transmembrane beta-barrel and the single or multiple membrane spanning alpha-helical proteins. They are imported via the SAM beta-barrel and alpha-helical import pathways using SAM and MIM systems, respectively. IMM can be subdivided into the inner boundary membrane and the cristae. The inner boundary membrane establishes contacts with OMM; the large invaginations of cristae protrude into the matrix. The two domains are connected by crista junctions. Complexes of OXPHOS are enriched in the cristae; the ATP synthase dimers are located at the tip of cristae. The large MINOS protein complex at the crista junction plays important roles in the elaborate architecture of IMM. MINOS interacts TOM and SAM complexes, transiently with MIA and with cardiolipin (CL). These interactions are likely involved in promoting translocation of precursaors into the intermembrane space on one hand, and in maintaining the integrity of crista junctions. OMM resident proteins are involved in apoptosis, mitochondrial autophagy and dynamics. Yet, the import of these proteins, particularly the alpha-helical ones, are not well understood and characterized, especially in higher eukaryotes. The import pathways have been most studied and are better characterized in yeast, though many molecular details continue to be elusive. Details of the individual import pathways, as known/applicable in higher eukaryotes, are presented.

Presequence pathway of mitochondrial protein import
The presequence pathway, regarded as the classical import pathway, is likely the most prevalent one. Many of the nuclear-encoded mitochondrial proteins have a cleavable, N-terminal presequence of 15-50 residues that forms a positively charged amphipathic helix. Presequence and many other precursors are recognized by Tomm20, 22 and 70 receptors of the OMM translocase TOM complex. Tom20 recognizes the hydrophobic site of the presequence helix, Tom22 the hydrophilic site. Tom22 has both a cytosolic and an intermembrane space exposed domain. The beta-barrel Tomm40 component constitutes the cargo-conducting channel. The small Tomm5, 6 and 7 subunits confer further support and/or stability. TOM activity is regulated via phosphorylation, both positively and negatively. TIM23 translocase mediates presequence protein transport into the matrix or lateral release into the IMM in a membrane-potential dependent manner. It contains the Timm23 channel, Timm50 main receptor and Timm21; their exposed soluble domains in the intermembrane space contact Tomm22 component of TOM. Two more elements are Timm17a and ROMO1 homolog of yeast Mgr2. All TIM23 proteins are membrane-embedded. Protein import into the matrix further requires the ATP-driven, translocase-associated motor PAM. An essential component of PAM is Hspa9 chaperone, known as the mitochondrial HSP70. Other components include the cochaperone Dnajc19, known as Pam18, Grpel1/2 known as Mge1 and the scaffolding Timm44 and Pam16 proteins. Timm44 is recruited to IMM via interactions with Timm23, Timm17a and cardiolipin, and in turns recruits the ATP-bound Hspa9. ATP hydrolysis, stimulated by Dnajc19, tightens the chaperone-substrate interaction and leads to dissociation from Timm44 and diffusion into the matrix. Dnajc19 recruitment to TIM23 is mediated by interactions with Timm17a and Pam16. Substrate release is facilitated by Grpel1/Grpel2 mediated ADP release. In the matrix the precursors are processed by mitochondrial processing peptidases alpha and beta, with additional processing by Xpnpep3, known as Icp55 or Mipep (Oct1 in yeast). Precursors delivered to IMM are processed primarily by the mitochondrial inter-membrane peptidases 1 and 2. Mitochondria-encoded proteins are inserted into IMM by the Oxa1l system. TIM and PAM systems are involved in complex IV assembly (cytochrome c oxidase). Many details of the complex network of interactions within and between the various complexes are still to be unraveled.

Carrier pathway of mitochondrial protein import
Carrier precursors such as the multispanning metabolite carriers, are synthesized without a cleavable presequence but contain several internal targeting signals. Tomm70 of TOM complex acts as the main receptor for the carrier precursors. As cargo proteins emerge from Tomm40 channel they are bound by members of the small Tim proteins. Five members in yeast and six in humans represent the small Tim members and while in both species they are important for delivery of carrier precursors to TIM22 translocase, they appear to have evolved different mechanisms. A hexameric complex of Timm9/Timm10 acts as a chaperone that escorts the cargo through the intermembrane space and delivers it to TIM22 translocase via the Tim9/Tim10/Tim12 complex in yeast. The equivalent human Timm9/Timm10 complex is not found in the intermembrane space. Human Timm9/Timm10 and Timm0/Timm10/Timm10b complexes are tightly associated with IMM. Tim proteins interact with the TIM22 pore component but they don¿t appear to exhibit the stable interaction seen in yeast and conferred by the Tim12 member. The twin-pore Timm22 is the essential component of TIM22 necessary for the membrane insertion of carrier precursors. The other TIM22 components, such as Tim18 and Tim54 in yeast, do not appear to be expressed in mammals. Tim proteins have four conserved cysteine residues in the twin CX3C motif forming a zinc-finger-like structure. In both lower and higher eukaryotes zinc is required for the import of carrier precursors. And in both, the pore activity is dependent upon IMM potential.

Intermembrane space assembly pathway of mitochondrial protein import
Cysteine-rich precursors destined for the intermembrane space (IMS) are imported via the mitochondrial IMS assembly (MIA) pathway. MIA40, official name CHCHD4, has been identified as the element responsible for the oxidative folding of substrates by promoting formation of disulfide bonds. Chchd4 is anchored to IMM via its hydrophobic N-terminus, its IMS domain contains six highly conserved cysteine residues, and the soluble C-terminus is exposed to the IMS. The six cysteines ¿ CPC; CX9C; CX9C ¿ have distinct functions. The cysteine-proline-cysteine (CPC) motif provides a small redox center: the second C catalyzes the formation of a mixed disulfide intermediate with incoming precursors that is finished by the first C. The other four cysteines form two intramolecular disulfide bridges that promote the structural integrity of Chchd4. After release of the precursor, Chchd4 must be reoxidized ¿ a function supplied by Erv1, official name Gfer. The mammalian GFER protein is the functional and structural homolog of yeast Erv1, a FAD-dependent essential sulfhydryl oxidase. The electron receiver for Gfer is cytochrome c oxidase (complex IV) of the OXPHOS system. While the cysteine-rich precursors are imported via TOM complex, this is independent of TOM receptors and relies on Tomm40 channel.

Beta-barrel pathway of mitochondrial protein import
The beta-barrel precursors that reside in the OMM employ the SAM system, coupled to TOM complex. First, they are recognized by Tom receptors that imports them into the IMS from where they are sorted and assembled into OMM by the SAM complex. The small Tim proteins Timm9 and Timm10a, and also Timm8 and Timm13, chaperone the precursors between TOM and SAM. TOM and SAM form a supercomplex viaTom22 component of TOM. SAM consists of three core components. Samm50 is the main core component, itself a beta-barrel protein composed of 16 beta-strands forming the channel and of a conserved polypeptide-transport associated domain (POTRA). The other two components are yeast Sam37 and 35 whose proposed mammalian homologs are metaxin1 and 2, respectively. Yeast complex contains an auxiliary component, Mdm10 which is part of larger structure (ERMES) tethering mitochondria to endoplasmic reticulum (ER). Mammalian counterparts of Mdm10 or of the other components of ERMES, if any, remain to be identified. Samm50 via its POTRA domain interacts with the MINOS complex. The yeast complex consists of six subunits; five are identified in higher eukaryotes.

alpha-helical insertion pathway of mitochondrial protein import
The alpha-helical proteins that reside in the OMM can have single or multiple membrane spanning helices and the internal target on the transmembrane domain (TMD) of single-spanning proteins can be located at the TMD N- or C-termini, or internally. Largely, the alpha-spanning proteins employ the MIM system. The precursors are recognized by Tomm70 receptor of the TOM complex. Subsequently, the precursors are transferred to the MIM complex which mediates their integration into OMM. The Mim1 and Mim2 yeast proteins do not have known homologs in higher eukaryotes. TOM, SAM and even TIM23 systems may have a role in the import of alpha-helical proteins. This is the least understood system and many aspects of its working in higher eukaryotes remain to be elucidated.

MINOS system
The mitochondrial inner-membrane organizing system (MINOS) also known as the mitochondrial contact-site (MICOS) complex or the mitochondrial organizing structure (MitOS) is found at cristae junction and plays a central role in the maintenance of mitochondrial membrane architecture. It is composed of six subunits in yeast, where it has first been identified, and five in mammals. Its interactions with TOM, SAM and MIA and the importance it has on the translocation of precursor protein into the inter-membrane space, establish a dual role for the MINOS complex. These interactions also establish a dual role for some of the translocases in the maintenance of cristae integrity, as has been shown for SAM. The five components of mammalian systems are mitofilin (yeast Fcj1), MINOS1 (yeast Mio10), CHCHD3 and CHCHD6 (yeast Aim3),APOO (yeast Aim27) and APOOL (yeast Aim37). Mitofilin (IMMT) is the core component of the system, MINOS1 is a small transmembrane protein important for complex stability, CHCHD3 and CHCHD6 are twin CX9C proteins that face the intermembrane space, APOO and APOOL are related proteins of which APOOL binds cardiolipin specifically. The MINOS components establish interactions with each other and with other proteins and have been associated with a range of human diseases that include Parkinson's, cancer, Down syndrome and diabetic cardiomyopathy. Mitochondria damage and dysfunction are associated with a range of conditions including neurodegenerative diseases and with aging.

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