Dissecting the interactions of PINK1 with the TOM complex in depolarized mitochondria

Mitochondria dysfunction is involved in the pathomechanism of many illnesses including Parkinson's disease. PINK1, which is mutated in some cases of familiar Parkinsonism, is a key component in the degradation of damaged mitochondria by mitophagy. The accumulation of PINK1 on the mitochondrial outer membrane (MOM) of compromised organelles is crucial for the induction of mitophagy, but the molecular mechanism of this process is still unresolved. Here, we investigate the association of PINK1 with the TOM complex. We demonstrate that PINK1 heavily relies on the import receptor TOM70 for its association with mitochondria and directly interacts with this receptor. The structural protein TOM7 appears to play only a moderate role in PINK1 association with the TOM complex, probably due to its role in stabilizing this complex. PINK1 requires the TOM40 pore lumen for its stable interaction with the TOM complex and apparently remains there during its further association with the MOM. Overall, this study provides new insights on the role of the individual TOM subunits in the association of PINK1 with the MOM of depolarized mitochondria.

Protein Quality Control at the Mitochondrial Surface

Mitochondria contain two membranes, the outer and inner membrane. The outer membrane fulfills crucial functions for the communication of mitochondria with the cellular environment like exchange of lipids via organelle contact sites, the transport of metabolites and the formation of a signaling platform in apoptosis and innate immunity. The translocase of the outer membrane (TOM complex) forms the entry gate for the vast majority of precursor proteins that are produced on cytosolic ribosomes. Surveillance of the functionality of outer membrane proteins is critical for mitochondrial functions and biogenesis. Quality control mechanisms remove defective and mistargeted proteins from the outer membrane as well as precursor proteins that clog the TOM complex. Selective degradation of single proteins is also an important mode to regulate mitochondrial dynamics and initiation of mitophagy pathways. Whereas inner mitochondrial compartments are equipped with specific proteases, the ubiquitin-proteasome system is a central player in protein surveillance on the mitochondrial surface. In this review, we summarize our current knowledge about the molecular mechanisms that govern quality control of proteins at the outer mitochondrial membrane.

Structure of PINK1 reveals autophosphorylation dimer and provides insights into binding to the TOM complex

Mutations in PINK1 causes autosomal-recessive Parkinson's disease. Mitochondrial damage results in PINK1 import arrest on the Translocase of the Outer Mitochondrial Membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.

The receptor subunit Tom20 is dynamically associated with the TOM complex in mitochondria of human cells

The outer membrane translocase (TOM) is the import channel for nuclear-encoded mitochondrial proteins. The general import pore contains Tom40, Tom22, Tom5, Tom6 and Tom7. Precursor proteins are bound by the peripheral receptor proteins Tom20, Tom22 and Tom70 before being imported by the TOM complex. Here we investigated the association of the receptor Tom20 with the TOM complex. Tom20 was found in the TOM complex, but not in a smaller subcomplex. In addition, a subcomplex was found without Tom40 and Tom7 but with Tom20. Using single particle tracking of labeled Tom20 in overexpressing human cells, we show that Tom20 has, on average, higher lateral mobility in the membrane than Tom7/TOM. After ligation of Tom20 with the TOM complex by post-tranlational protein trans-splicing using the trackless, ultra-fast cleaved Gp41-1 integrin system, a significant decrease in the mean diffusion coefficient of Tom20 was observed in the resulting Tom20-Tom7 fusion protein. Exposure of Tom20 to high substrate loading also resulted in reduced mobility. Taken together, our data show that the receptor subunit Tom20 interacts dynamically with the TOM core complex. We suggest that the TOM complex containing Tom20 is the active import pore and that Tom20 is associated when substrate is available.

A Biochemical and Structural Understanding of TOM Complex Interactions and Implications for Human Health and Disease

The central role mitochondria play in cellular homeostasis has made its study critical to our understanding of various aspects of human health and disease. Mitochondria rely on the translocase of the outer membrane (TOM) complex for the bulk of mitochondrial protein import. In addition to its role as the major entry point for mitochondrial proteins, the TOM complex serves as an entry pathway for viral proteins. TOM complex subunits also participate in a host of interactions that have been studied extensively for their function in neurodegenerative diseases, cardiovascular diseases, innate immunity, cancer, metabolism, mitophagy and autophagy. Recent advances in our structural understanding of the TOM complex and the protein import machinery of the outer mitochondrial membrane have made structure-based therapeutics targeting outer mitochondrial membrane proteins during mitochondrial dysfunction an exciting prospect. Here, we describe advances in understanding the TOM complex, the interactome of the TOM complex subunits, the implications for the development of therapeutics, and our understanding of the structure/function relationship between components of the TOM complex and mitochondrial homeostasis.

Localized Proteotoxic Stress in Mitochondrial Intermembrane Space and Matrix Elicits Sub-compartment Specific Response Pathways Governed by Unique Modulators

The complex double-membrane architecture of mitochondria is essential for its ATP synthesis function and divides the organelle into two sub-mitochondrial compartments, inter-membrane space (IMS) and matrix. The folding environments of IMS and matrix are significantly different owing to its dissimilar oxido-reductive environments and distinctly divergent protein quality control (PQC) machineries. Here, by inducing proteotoxic stress restricted to IMS or matrix by targeting three different stressor proteins, we show that the cellular response to IMS or matrix-localized misfolding stress is distinct and unique. IMS and matrix stress response pathways are quite effective in combatting stress despite significant stress-induced alteration in mitochondrial phenotypes. IMS misfolding stress leads to specific upregulation of IMS chaperones and components of TOM complex while matrix chaperones and cytosolic PQC components are upregulated during matrix stress. Notably, the amplitude of upregulation of mitochondrial chaperones is not overwhelming. We report that cells respond to mitochondrial stress through an adaptive mechanism by adjourning mitochondrial respiration while upregulating glycolysis as a compensatory pathway. We show that subunits of TOM complex act as specific modulators of IMS-stress response while Vms1 precisely modulates the matrix stress response.

USP30 sets a trigger threshold for PINK1–PARKIN amplification of mitochondrial ubiquitylation

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition.

From TOM to the TIM23 complex – handing over of a precursor

Mitochondrial precursor proteins with amino-terminal presequences are imported via the presequence pathway, utilizing the TIM23 complex for inner membrane translocation. Initially, the precursors pass the outer membrane through the TOM complex and are handed over to the TIM23 complex where they are sorted into the inner membrane or translocated into the matrix. This handover process depends on the receptor proteins at the inner membrane, Tim50 and Tim23, which are critical for efficient import. In this review, we summarize key findings that shaped the current concepts of protein translocation along the presequence import pathway, with a particular focus on the precursor handover process from TOM to the TIM23 complex. In addition, we discuss functions of the human TIM23 pathway and the recently uncovered pathogenic mutations in TIM50.