Mitochondrial Protein Import Pathways
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Mitochondrial Protein Import Pathways

Eukaryotic cells are characterized by extensive subcellular compartmentation whose structural basis is the existence of a number of highly specialized membrane-bound organelles. Each of these organelles is equipped with a specific subset of proteins allowing them to fulfill specific tasks in cellular metabolism. Mitochondria are present in virtually all eukaryotic cells. Mitochondria are the compartments responsible for respiration and oxidative phosphorlyation. They are made up of two highly specialized membrane systems, the OM (Outer Membrane) and IM (Inner Membrane), and two aqueous compartments, the Matrix and the IMS (Intermembrane Space). More than 90% of the mitochondrial proteins are encoded on the nuclear DNA and then synthesized as precursors on cytosolic ribosomes after which they must be imported into the organelle. The mitochondrial membranes contain specific machineries (translocases) for recognition, translocation, and membrane insertion of precursor proteins. Mitochondrial precursor proteins can be separated into two main classes. Preproteins that are destined for the mitochondrial matrix, as well as a number of proteins of the inner membrane and intermembrane space, carry N-terminal cleavable extensions, termed presequences. These positively charged extensions function as targeting signals that interact with the mitochondrial import receptors and direct the preproteins across both outer and inner membranes. The second class of precursor proteins, carrying various internal targeting signals, includes all outer membrane proteins along with many intermembrane space and inner membrane proteins. These precursors are synthesized without cleavable extensions, that is, they have the same primary structure as the mature protein, yet their conformation typically differs from the mature protein (Ref.1 & 2).

The mitochondrial preproteins are believed to be maintained in a translocation competent state by cytosolic Chaperones, in particular members of the HSP70 (Heat shock protein family of 70 kDa) protein family and by binding factors specific for presequences. The translocation across the mitochondrial membranes is then mediated by the import machineries of the Outer (TOM (Translocase of Outer Mitochondrial membrane) Complex) and the inner membrane (TIM (Translocase of Inner Mitochondrial Membrane) Complex) at sites of close contact between both membranes. Receptors on the outer surface of the mitochondrial outer membrane specifically recognize and bind the precursors prior to their translocation. The translocase of the outer mitochondrial membrane (TOM Complex) represents the central entry gate for practically all nuclear-encoded mitochondrial proteins. The TOM complex consists of seven different subunits that can be grouped into three categories: the receptors TOM20, TOM22, and TOM70; the channel-forming protein TOM40; and three small TOM proteins, TOM5, TOM6, and TOM7. TOM20 is the first receptor involved in recognizing the presequence of a preprotein. A typical presequence has a length of about 10-30 amino acid residues and forms an amphipathic Alpha-helix. One half of the helix possesses a hydrophobic surface that is recognized by a binding groove within TOM20, whereas the other half is positively charged and recognized by the receptor TOM22. With the help of the small protein TOM5, the preprotein is then transported to the general import pore formed by the essential Beta-barrel protein TOM40. After translocation through the TOM40 pore, the presequence binds to the intermembrane space domain of the receptor TOM22. TOM40 itself does not simply form a passive pore but rather interacts with the preproteins in transit. The other small TOM proteins, TOM6 and TOM7, do not directly interact with precursor proteins but are required for the assembly and stability of the TOM complex (Ref.3 & 4).

After passing through the TOM complex, the precursor proteins can follow one of three major pathways. Preproteins with a presequence are transferred to the presequence translocase of the inner membrane, also termed the TIM23 complex (translocase of the inner membrane. The presequence translocase of the inner membrane consists of three integral and essential membrane proteins: TIM50, TIM23, and TIM17. After release from the TOM complex, the presequence-carrying preproteins first contact TIM50, which exposes a large domain to the intermembrane space. TIM50 then guides the preproteins to the import channel formed by TIM23. TIM17 is tightly associated with TIM23 and probably influences the channel activity, although the exact role of TIM17 remains unknown. Insertion of preproteins into the TIM23 channel strictly depends on the presence of the membrane potential across the inner membrane. The membrane potential plays a dual role in the import process. It activates the channel protein itself as well as exert an electrophoretic effect on the positively charged presequences, thereby driving the presequences to the matrix side. The presequence translocase forms a channel across the inner membrane and cooperates with the mtHSP70 (matrix Heat Shock Protein-70). The molecular Chaperone mtHSP70 represents the core of the PAM (Presequence Translocase-Associated Motor) Complex, which drives the completion of protein transport into the matrix. Besides mtHSP70, PAM also consists of the peripheral inner membrane protein TIM44, which binds mtHSP70 and directs it to the TIM23 complex; and the nucleotide exchange factor of the matrix, termed mitochondrial GrpE (Mge1). Recent studies have led to the identification of two more membrane-bound essential Co-chaperones, PAM18/TIM14 and PAM16/ TIM16. PAM18 belongs to the J-protein family of Co-chaperones and stimulates the ATPase activity of mtHSP70. PAM16 is involved in the recruitment of PAM18 to the TIM23 complex. The molecular mechanism of the import motor probably involves both passive trapping and active pulling of the preprotein. When bound to ATP, mtHSP70 interacts with TIM44 and preproteins with a relatively low affinity. The hydrolysis of ATP stimulated by PAM18 leads to the ADP-bound form of mtHSP70 and thus stabilizes its interaction with TIM44 and preproteins. The soluble matrix protein GrpE1 then promotes the release of ADP from mtHSP70. Thus, the PAM operates as a multistep motor, although the exact order of events driving the reaction cycle of mtHSP70, including binding and release of preproteins, is not fully understood. Independently from PAM involvement, the MPP (Mitochondrial Processing Peptidase) removes the presequences from the preproteins. Another Chaperone (called a Chaperonin), HSP60 (Heat Shock Protein-60) causes the folding of the protein into its tertiary sequence. This process also utilizes an ATP molecule (Ref.5, 6 & 7).

Unlike Preproteins with a presequence, the Outer membrane preproteins and Carrier preproteins follow different routes after passing through the TOM complex. After transport through the TOM machinery, the SAM (Sorting and Assembly Machinery complex) Complex is required for the insertion of Beta-barrel Outer membrane proteins into the Outer membrane. The precursors of the hydrophobic Carrier proteins of the inner membrane, e.g. the ADP/ATP carrier or the Phosphate carrier, are initially recognized by a different receptor at the mitochondrial surface, TOM70. These precursors are usually synthesized without a presequence but contain multiple internal targeting signals. After recognition by TOM70 and translocation by the TOM pore across the outer membrane, these Carrier proteins are bound by a complex of small TIM proteins in the intermembrane space, the TIM9-TIM10 complex. Besides the essential TIM9-TIM10 complex, the homologous yet non-essential TIM8-TIM13 complex also supports the transfer of selected inner membrane proteins. The TIM9-TIM10 complex delivers the precursor proteins to the membrane-integrated insertion machinery of the inner membrane by directly docking to it. This insertion machinery is termed the TIM22 complex or the carrier translocase. The peripheral membrane protein TIM12 serves as the docking site for the TIM9-TIM10 complex. The precursor is then translocated to the core of the insertion machinery, the channel-forming protein TIM22. The functions of two further subunits of the carrier translocase, the membrane proteins TIM18 and TIM54, are not yet defined (Ref. 8 & 9).

Thus, mitochondrial import requires the action of a variety of components in the cytosol to maintain an import-competent conformation, in the outer mitochondrial membrane for recognition of the precursor, in both outer and inner membrane for protein translocation, and in the matrix for proteolytic processing and folding to the active conformation. Any defect in any of the above processes may lead to mitochondrial disorders like Deafness-Dystonia-Dementia Syndrome, Mohr-Tranebjaerg syndrome and Jensen syndrome. (Ref.1 & 10).