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.

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Functions of the Small Proteins in the TOM Complex of Neurospora crasssa

Molecular Biology of the Cell ◽

10.1091/mbc.e05-03-0187 ◽

2005 ◽

Vol 16 (9) ◽

pp. 4172-4182 ◽

Cited By ~ 50

Author(s):

E. Laura Sherman ◽

Nancy E. Go ◽

Frank E. Nargang

Keyword(s):

Mitochondrial Membrane ◽

Minor Role ◽

Complex Stability ◽

Outer Mitochondrial Membrane ◽

The Core ◽

Tom Complex ◽

Membrane Complex ◽

Small Proteins ◽

Precursor Proteins ◽

A Minor

The TOM (translocase of the outer mitochondrial membrane) complex of the outer mitochondrial membrane is required for the import of proteins into the organelle. The core TOM complex contains five proteins, including three small components Tom7, Tom6, and Tom5. We have created single and double mutants of all combinations of the three small Tom proteins of Neurospora crassa. Analysis of the mutants revealed that Tom6 plays a major role in TOM complex stability, whereas Tom7 has a lesser role. Mutants lacking both Tom6 and Tom7 have an extremely labile TOM complex and are the only class of mutant to exhibit an altered growth phenotype. Although single mutants lacking N. crassa Tom5 have no apparent TOM complex abnormalities, studies of double mutants lacking Tom5 suggest that it also has a minor role in maintaining TOM complex stability. Our inability to isolate triple mutants supports the idea that the three proteins have overlapping functions. Mitochondria lacking either Tom6 or Tom7 are differentially affected in their ability to import different precursor proteins into the organelle, suggesting that they may play roles in the sorting of proteins to different mitochondrial subcompartments. Newly imported Tom40 was readily assembled into the TOM complex in mitochondria lacking any of the small Tom proteins.

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A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria

Molecular Biology of the Cell ◽

10.1091/mbc.e14-06-1155 ◽

2014 ◽

Vol 25 (25) ◽

pp. 3999-4009 ◽

Cited By ~ 23

Author(s):

Agnieszka Gornicka ◽

Piotr Bragoszewski ◽

Piotr Chroscicki ◽

Lena-Sophie Wenz ◽

Christian Schulz ◽

...

Keyword(s):

Outer Membrane ◽

Mitochondrial Membrane ◽

Alternative Pathway ◽

Intermembrane Space ◽

Outer Mitochondrial Membrane ◽

Membrane Translocation ◽

Tom Complex ◽

Precursor Proteins ◽

Core Components

Mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria with the help of protein translocases. For the majority of precursor proteins, the role of the translocase of the outer membrane (TOM) and mechanisms of their transport across the outer mitochondrial membrane are well recognized. However, little is known about the mode of membrane translocation for proteins that are targeted to the intermembrane space via the redox-driven mitochondrial intermembrane space import and assembly (MIA) pathway. On the basis of the results obtained from an in organello competition import assay, we hypothesized that MIA-dependent precursor proteins use an alternative pathway to cross the outer mitochondrial membrane. Here we demonstrate that this alternative pathway involves the protein channel formed by Tom40. We sought a translocation intermediate by expressing tagged versions of MIA-dependent proteins in vivo. We identified a transient interaction between our model substrates and Tom40. Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22. Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

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Mitochondrial Protein Import

Journal of Biological Chemistry ◽

10.1074/jbc.m404591200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45701-45707 ◽

Cited By ~ 45

Author(s):

Masatoshi Esaki ◽

Hidaka Shimizu ◽

Tomoko Ono ◽

Hayashi Yamamoto ◽

Takashi Kanamori ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Protein Import ◽

Protein Translocation ◽

Mitochondrial Protein ◽

Intermembrane Space ◽

Outer Mitochondrial Membrane ◽

Mitochondrial Protein Import ◽

Tom Complex ◽

Membrane Complex ◽

Cytosolic Side

Protein translocation across the outer mitochondrial membrane is mediated by the translocator called the TOM (translocase of the outer mitochondrial membrane) complex. The TOM complex possesses two presequence binding sites on the cytosolic side (thecissite) and on the intermembrane space side (thetranssite). Here we analyzed the requirement of presequence elements and subunits of the TOM complex for presequence binding to thecisandtranssites of the TOM complex. The N-terminal 14 residues of the presequence of subunit 9 of F0-ATPase are required for binding to thetranssite. The interaction between the presequence and thecissite is not sufficient to anchor the precursor protein to the TOM complex. Tom7 constitutes or is close to thetranssite and has overlapping functions with the C-terminal intermembrane space domain of Tom22 in the mitochondrial protein import.

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A novel USP30 inhibitor recapitulates genetic loss of USP30 and sets the trigger for PINK1-PARKIN amplification of mitochondrial ubiquitylation

10.1101/2020.04.16.044206 ◽

2020 ◽

Author(s):

Emma Rusilowicz-Jones ◽

Jane Jardine ◽

Andreas Kallinos ◽

Adan Pinto-Fernandez ◽

Franziska Guenther ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Neuroblastoma Cell ◽

Selective Inhibition ◽

Neuroblastoma Cell Line ◽

Outer Mitochondrial Membrane ◽

Loss Of Function ◽

Mitochondrial Mass ◽

Tom Complex ◽

Membrane Depolarisation ◽

Disease Associated Genes

AbstractThe mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinson’s Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. 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 most 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. In this model, 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 and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.

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How does the TOM complex mediate insertion of precursor proteins into the mitochondrial outer membrane?

The Journal of Cell Biology ◽

10.1083/jcb.200507147 ◽

2005 ◽

Vol 171 (3) ◽

pp. 419-423 ◽

Cited By ~ 72

Author(s):

Doron Rapaport

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Mitochondrial Membrane ◽

Mitochondrial Outer Membrane ◽

Outer Face ◽

Outer Mitochondrial Membrane ◽

Lipid Core ◽

Tom Complex ◽

Precursor Proteins

A multisubunit translocase of the outer mitochondrial membrane (TOM complex) mediates both the import of mitochondrial precursor proteins into the internal compartments of the organelle and the insertion of proteins residing in the mitochondrial outer membrane. The proposed β-barrel structure of Tom40, the pore-forming component of the translocase, raises the question of how the apparent uninterrupted β-barrel topology can be compatible with a role of Tom40 in releasing membrane proteins into the lipid core of the bilayer. In this review, I discuss insertion mechanisms of proteins into the outer membrane and present alternative models based on the opening of a multisubunit β-barrel TOM structure or on the interaction of outer membrane precursors with the outer face of the Tom40 β-barrel structure.

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

Cells ◽

10.3390/cells10051164 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1164

Author(s):

Ashley S. Pitt ◽

Susan K. Buchanan

Keyword(s):

Human Health ◽

Mitochondrial Membrane ◽

Cancer Metabolism ◽

Protein Import ◽

Mitochondrial Protein ◽

Outer Mitochondrial Membrane ◽

Tom Complex ◽

Complex Interactions ◽

Health And Disease ◽

Function Relationship

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.

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Wild-type and mutant (G2019S) leucine-rich repeat kinase 2 (LRRK2) associate with subunits of the translocase of outer mitochondrial membrane (TOM) complex

Experimental Cell Research ◽

10.1016/j.yexcr.2018.12.022 ◽

2019 ◽

Vol 375 (2) ◽

pp. 72-79 ◽

Cited By ~ 1

Author(s):

Annika Neethling ◽

Lize Engelbrecht ◽

Ben Loos ◽

Craig Kinnear ◽

Rensu Theart ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Outer Mitochondrial Membrane ◽

Leucine Rich Repeat ◽

Wild Type ◽

Tom Complex

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

Life Science Alliance ◽

10.26508/lsa.202000768 ◽

2020 ◽

Vol 3 (8) ◽

pp. e202000768 ◽

Cited By ~ 4

Author(s):

Emma V Rusilowicz-Jones ◽

Jane Jardine ◽

Andreas Kallinos ◽

Adan Pinto-Fernandez ◽

Franziska Guenther ◽

...

Keyword(s):

Mitochondrial Membrane ◽

High Selectivity ◽

Neuroblastoma Cell ◽

Neuroblastoma Cell Line ◽

Outer Mitochondrial Membrane ◽

Proteomics Analysis ◽

Tom Complex ◽

Cell Line Model ◽

Membrane Depolarisation ◽

Chemical Inhibition

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.

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Biogenesis of the preprotein translocase of the outer mitochondrial membrane: protein kinase A phosphorylates the precursor of Tom40 and impairs its import

Molecular Biology of the Cell ◽

10.1091/mbc.e11-11-0933 ◽

2012 ◽

Vol 23 (9) ◽

pp. 1618-1627 ◽

Cited By ~ 53

Author(s):

Sanjana Rao ◽

Oliver Schmidt ◽

Angelika B. Harbauer ◽

Birgit Schönfisch ◽

Bernard Guiard ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Mitochondrial Membrane ◽

Inhibitory Effect ◽

Outer Mitochondrial Membrane ◽

Central Channel ◽

Receptor Function ◽

Tom Complex ◽

Mitochondrial Membrane Protein ◽

Kinase A

The preprotein translocase of the outer mitochondrial membrane (TOM) functions as the main entry gate for the import of nuclear-encoded proteins into mitochondria. The major subunits of the TOM complex are the three receptors Tom20, Tom22, and Tom70 and the central channel-forming protein Tom40. Cytosolic kinases have been shown to regulate the biogenesis and activity of the Tom receptors. Casein kinase 2 stimulates the biogenesis of Tom22 and Tom20, whereas protein kinase A (PKA) impairs the receptor function of Tom70. Here we report that PKA exerts an inhibitory effect on the biogenesis of the β-barrel protein Tom40. Tom40 is synthesized as precursor on cytosolic ribosomes and subsequently imported into mitochondria. We show that PKA phosphorylates the precursor of Tom40. The phosphorylated Tom40 precursor is impaired in import into mitochondria, whereas the nonphosphorylated precursor is efficiently imported. We conclude that PKA plays a dual role in the regulation of the TOM complex. Phosphorylation by PKA not only impairs the receptor activity of Tom70, but it also inhibits the biogenesis of the channel protein Tom40.

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