The Journey of Mitochondrial Protein Import and the Roadmap to Follow

Abstract

Mitochondria are double membrane-bound organelles that play critical functions in cells including metabolism, energy production, regulation of intrinsic apoptosis, and maintenance of calcium homeostasis. Mitochondria are fascinatingly equipped with their own genome and machinery for transcribing and translating 13 essential proteins of the oxidative phosphorylation system (OXPHOS). The rest of the proteins (99%) that function in mitochondria in the various pathways described above are nuclear-transcribed and synthesized as precursors in the cytosol. These proteins are imported into the mitochondria by the unique mitochondrial protein import system that consists of seven machineries. Proper functioning of the mitochondrial protein import system is crucial for optimal mitochondrial deliverables, as well as mitochondrial and cellular homeostasis. Impaired mitochondrial protein import leads to proteotoxic stress in both mitochondria and cytosol, inducing mitochondrial unfolded protein response (UPR^mt). Altered UPR^mt is associated with the development of various disease conditions including neurodegenerative and cardiovascular diseases, as well as cancer. This review sheds light on the molecular mechanisms underlying the import of nuclear-encoded mitochondrial proteins, the consequences of defective mitochondrial protein import, and the pathological conditions that arise due to altered UPR^mt.

Keywords: diseases; mitochondria; mitochondrial protein import machineries; mitochondrial unfolded protein response; proteins.

Figure 1

The mitochondrial protein import system. The mitochondrial protein import system consists of seven machineries, including the translocase of the outer mitochondrial membrane (TOMM) machinery, mitochondrial import machinery (MIM), sorting and assembly machinery (SAM), mitochondrial intermembrane space import and assembly machinery (MIA), translocase of the inner mitochondrial membrane 23 (TIMM 23) machinery, translocase of the inner mitochondrial membrane 22 (TIMM 22) machinery, and a presequence-associated motor (PAM). The figure was created with Biorender.com.

Figure 2

Import of β-barrel precursors into the outer mitochondrial membrane. (A) Upon translocation through the TOMM40 channel, the precursors bind to TIMM9–TIMM10 complex, which protect the β-barrel precursors from aggregation in the aqueous IMS and deliver the β-barrel precursors to the SAM complex. Subsequently, the precursors are folded in the SAM complex and laterally released into the lipid phase of the outer membrane. (B) SAM37 interacts with the cytosolic receptor domain of TOMM22, thereby linking the two complexes and leading to the formation of a TOMM–SAM supercomplex, which enables the binding of SAM35 to the β signal of the precursor, thereby allowing the direct transfer of the β-barrel precursors from TOMM to the SAM complex. Subsequently, the β-barrel precursors are inserted into the SAM50 channel, after which they are folded in the SAM complex and released laterally into the lipid phase of the outer membrane. IMS, intermembrane space. The figure was created with Biorender.com.

Figure 3

Import of α-helical precursors into the outer mitochondrial membrane. Polytopic proteins are recognized by the TOMM70 receptor, after which TOMM70 binds to them and transfers them to the MIM complex, which inserts them into the OMM. Signal- and tail-anchored α-helical precursors are also inserted into the OMM by the MIM complex. The exact TOMM receptors recognizing these precursors have not been identified yet. OMM, outer mitochondrial membrane. The figure was created with Biorender.com.

Figure 4

Import of intermembrane space proteins. As the precursors pass through the TOMM40 channel, the IMS sorting signals of these precursors are recognized by MIA40. Thereafter, MIA40 binds the precursors and facilitates their entry into the IMS. The imported precursors are oxidized by the oxidoreductase activity of MIA40, after which they are assembled in the IMS. In turn, MIA40 becomes reduced and is reoxidized by Erv1/ALR with the assistance of the zinc-binding protein–Helper of Tim protein 13 (Hot13). Electrons derived from the oxidation of the imported precursors by MIA40 are transferred to Erv1/ALR and, subsequently, to cytochrome C and complex IV. IMS, intermembrane space; Erv1, essential for respiration and viability 1 protein; ALR, augmenter of liver regeneration. The figure was created with Biorender.com.

Figure 5

Import of inner-mitochondrial membrane and matrix proteins. The presequence-carrying precursors are first recognized by TOMM20, which binds to them and transfers them to TOMM22, after which they are translocated through the TOMM40 channel, through which they enter the IMS, where they bind to the IMS domain of TOMM22. TIMM21 then binds the IMS domain of TOMM22, thereby promoting the dissociation of the precursors. Thereafter, TIMM50 binds to the precursor proteins and transfers the precursors into the TIMM23 channel. The Δψm exerts an electrophoretic effect on the positively charged N terminus of precursors and activates the TIMM23 channel, thereby aiding in the movement of the precursors through the TIMM23 channel. The hydrophobic sorting signals of the precursors are then recognized by Mgr2, which then binds to the sorting signals and controls the release of the precursors into the inner membrane. Subsequently, the inner-membrane peptidase (IMP) removes the hydrophobic sorting sequences, and the mature proteins are either released into the IMS or remain anchored in the inner membrane by an additional hydrophobic segment. Precursor proteins containing presequences devoid of hydrophobic sorting signals are destined for the matrix and are imported through the cooperation of the TOMM, TIMM23^CORE, and PAM machineries. After translocation through the TOMM40 channel, these precursors bind to the IMS domain of TOMM22, after which TIMM50 binds these precursors and transfers them to the TIMM23 channel. TIMM44 then binds to the precursor as it emerges on the matrix side of the TIMM23 channel and transfers it to mtHsp70, which imports the protein into the matrix. The presequences are removed by the matrix processing peptidase (MMP), and the proteins are folded into their mature forms by the soluble form of mtHSP70 and the HSP60-HSP10 chaperonin complex. Oxa1 aids in the export of some of the transmembrane segments of some inner-membrane precursors from the matrix into the inner mitochondrial membrane. Following their synthesis in the cytosol, carrier precursors are bound to cytosolic chaperones of the Hsp70 and Hsp90 classes to prevent aggregation. Thereafter, these chaperones deliver the precursors to TOMM70, which then transfers this precursor to TOMM22, after which they are transferred to the TOMM40 channel and translocated across the OMM in a loop formation. The small TIM chaperones of the IMS are recruited to the channel exit by an N-terminal segment of the channel protein TOMM40. TIMM54, a subunit of the TIMM22 machinery, recruits the small TIMM chaperones to the TIMM22 complex, after which, the precursors are delivered to the TIMM22 channel. The Δψm activates the TIMM22 channel and exerts an electrophoretic effect on the carrier precursors, which aids in the movement of the precursors through the channel. Finally, the precursors are released laterally from the TIMM22 complex into the IMM. OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane, IMS, intermembrane space; Δψm, mitochondrial membrane potential. The figure was created with Biorender.com.