Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex

Transport of nuclear encoded proteins into mitochondria is mediated by multisubunit translocation machineries in the outer and inner membranes of mitochondria. The TOM complex contains receptor and pore components that facilitate the recognition of preproteins and their transfer through the outer membrane. In addition, the complex contains a set of small proteins. Tom7 and Tom6 have been found in Neurospora and yeast, Tom5 has been found so far only in the latter organism. In the present study, we identified Neurospora Tom5 and analyzed its function in comparison to yeast Tom5, which has been proposed to play a role as a receptor-like component. Neurospora Tom5 crosses the outer membrane with its carboxyl terminus facing the intermembrane space like the other small Tom components. The temperature-sensitive growth phenotype of the yeast TOM5 deletion was rescued by overexpression of Neurospora Tom5. On the other hand, Neurospora cells deficient in tom5 did not exhibit any defect in growth. The structural stability of TOM complexes from cells devoid of Tom5 was significantly altered in yeast but not in Neurospora. The efficiency of protein import in Neurospora mitochondria was not affected by deletion of tom5, whereas in yeast it was reduced as compared with wild type. We conclude that the main role of Tom5, rather than being a receptor, is maintaining the structural integrity of the TOM complex.

Section: Discussionsupporting

confidence: 55%

“…Studies on the isolated TOM complex from Neurospora crassa and Saccharomyces cerevisiae have provided detailed information about the structure and function of this translocase (7)(8)(9)(10)(11)(12). The holocomplex consists of two receptor subunits, Tom70 and Tom20, and the proteins Tom40, Tom22, Tom7, and Tom6.…”

mentioning

confidence: 99%

Role of Tom5 in Maintaining the Structural Stability of the TOM Complex of Mitochondria

Schmitt

Ahting²,

Eichacker

et al. 2005

“…NMR, on the other hand, would be an alternative, and work is underway to obtain high resolution information. Electron microscopy has been used to obtain low resolution structures for the TIM22 and TOM40 complexes (25)(26)(27)(28), but the size of the TIM10 complex (70 kDa) is rather small, hindering the application of this method. Therefore, we obtained the low resolution three-dimensional structure of the individual proteins and their complex from SAXS experiments (Fig.…”

Section: Wtmentioning

confidence: 99%

The Structural Basis of the TIM10 Chaperone Assembly

Golovanov

Alcock

et al. 2004

Tim9 and Tim10 are essential components of the "small Tim" family of proteins that facilitate insertion of polytopic proteins at the inner mitochondrial membrane. The small Tims are themselves imported from the cytosol and are organized in specific translocation assemblies in the intermembrane space. Their conformational properties and how these influence the mechanism of assembly remain poorly understood. Moreover, the three-dimensional structure of the TIM10 complex is unknown. We have characterized the structural properties of these proteins in their free and assembled states using NMR, circular dichroism, and small angle x-ray scattering. We show that the free proteins are largely unfolded in their reduced assembly-incompetent state and molten globules in their oxidized assembly-competent state. Tim10 appears less structured than Tim9 in their respective free oxidized forms and undergoes a larger structural change than Tim9 upon complexation. The NMR data here demonstrates unequivocally that only the oxidized states of the Tim9 and Tim10 proteins are capable of forming a complex. Zinc binding stabilizes the reduced state against proteolysis without significantly affecting the secondary structure. Solution x-ray scattering was used to obtain a molecular envelope for the subunits individually and for their fully functional TIM10 complex. Ab initio shape reconstructions based on the scattering data has allowed us to obtain the first low resolution three-dimensional structure of the TIM10 complex. This is a novel structure that displays extensive surface hydrophobicity. The structure also provides an explanation for the escorting function of this non-ATP-powered chaperone particle.A universal event for the successful construction and function of all of the cells is that proteins are targeted to their correct location both spatially and temporally. The extent of these targeting events is reflected by the fact that more than one-third of the proteome of the cell are translocated across or inserted into a membrane. Mitochondria represent a major site for extensive protein translocation. Virtually all of the mitochondrial proteins (approximately one thousand different polypeptides) are imported from the cytosol and are sorted within the organelle. This event is orchestrated by distinct translocation machineries in the outer (translocase of the outer membrane, TOM) 1 and inner (translocase of the inner membrane, TIM) membranes and within the intermembrane space. An intriguing new class of translocase proteins is the small Tim family, which specifically mediates the import of presequencedevoid polytopic proteins.There are two main pathways for proteins destined to mitochondria. The matrix pathway is used mainly by precursors that contain a cleavable presequence (1), whereas precursors devoid of a presequence divert through the distinct carrier pathway (2-5). The small Tims are organized in two distinct 70-kDa complexes in the intermembrane space, the TIM10 complex that is made of Tim9 and Tim10, which are encoded by e...

“…A similar GIP complex containing Tom20 was also purified by affinity chromatography (20). Electron micrographs of the TOM complex isolated from wild-type yeast have shown three pores, whereas those from a yeast mutant lacking Tom20 showed only two pores (15,17,21,22). Furthermore, when the membrane potential was disrupted, the import of Tom70-dependent ADP/ATP carrier was arrested in the GIP complex, and interestingly, the ADP/ATP carrier generated a cross-link with Tom20, as well as Tom70 (23).…”

mentioning

confidence: 99%

Interaction between the Human Mitochondrial Import Receptors Tom20 and Tom70 in Vitro Suggests a Chaperone Displacement Mechanism

Fan

Kozlov

Hoegl

et al. 2011

The mitochondrial import receptor Tom70 contains a tetratricopeptide repeat (TPR) clamp domain, which allows the receptor to interact with the molecular chaperones, Hsc70/ Hsp70 and Hsp90. Preprotein recognition by Tom70, a critical step to initiate import, is dependent on these cytosolic chaperones. Preproteins are subsequently released from the receptor for translocation across the outer membrane, yet the mechanism of this step is unknown. Here, we report that Tom20 interacts with the TPR clamp domain of Tom70 via a conserved C-terminal DDVE motif. This interaction was observed by cross-linking endogenous proteins on the outer membrane of mitochondria from HeLa cells and in co-precipitation and NMR titrations with purified proteins. Upon mutation of the TPR clamp domain or deletion of the DDVE motif, the interaction was impaired. In co-precipitation experiments, the Tom20-Tom70 interaction was inhibited by C-terminal peptides from Tom20, as well as from Hsc70 and Hsp90. The Hsp90-Tom70 interaction was measured with surface plasmon resonance, and the same peptides inhibited the interaction. Thus, Tom20 competes with the chaperones for Tom70 binding. Interestingly, antibody blocking of Tom20 did not increase the efficiency of Tom70-dependent preprotein import; instead, it impaired the Tom70 import pathway in addition to the Tom20 pathway. The functional interaction between Tom20 and Tom70 may be required at a later step of the Tom70-mediated import, after chaperone docking. We suggest a novel model in which Tom20 binds Tom70 to facilitate preprotein release from the chaperones by competition.