Defining the architecture of the human TIM22 complex by chemical crosslinking

Anusha Valpadashi; Sylvie Callegari; Andreas Linden; Piotr Neumann; Ralf Ficner; Henning Urlaub; Markus Deckers; Peter Rehling

doi:10.1002/1873-3468.13978

Defining the architecture of the human TIM22 complex by chemical crosslinking

FEBS Lett. 2021 Jan;595(2):157-168. doi: 10.1002/1873-3468.13978. Epub 2020 Nov 13.

Authors

Anusha Valpadashi¹, Sylvie Callegari^{1

2

3}, Andreas Linden^{4

5}, Piotr Neumann⁶, Ralf Ficner^{6

7}, Henning Urlaub^{4

5}, Markus Deckers¹, Peter Rehling^{1

7

8}

Affiliations

¹ Department of Cellular Biochemistry, University Medical Center Göttingen, Germany.
² Ubiquitin Signalling Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.
³ Department of Medical Biology, University of Melbourne, Australia.
⁴ Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
⁵ Department of Clinical Chemistry, Bioanalytics, University Medical Center Göttingen, Germany.
⁶ Department of Molecular Structural Biology, Institute for Microbiology and Genetics, Göttingen Center for Molecular Biosciences, Georg-August-University Göttingen, Germany.
⁷ Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Goettingen, Germany.
⁸ Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

PMID: 33125709
DOI: 10.1002/1873-3468.13978

Abstract

The majority of mitochondrial proteins are nuclear encoded and imported into mitochondria as precursor proteins via dedicated translocases. The translocase of the inner membrane 22 (TIM22) is a multisubunit molecular machine specialized for the translocation of hydrophobic, multi-transmembrane-spanning proteins with internal targeting signals into the inner mitochondrial membrane. Here, we undertook a crosslinking-mass spectrometry (XL-MS) approach to determine the molecular arrangement of subunits of the human TIM22 complex. Crosslinking of the isolated TIM22 complex using the BS3 crosslinker resulted in the broad generation of crosslinks across the majority of TIM22 components, including the small TIM chaperone complex. The crosslinking data uncovered several unexpected features, opening new avenues for a deeper investigation into the steps required for TIM22-mediated translocation in humans.

Keywords: TIM22; carrier translocase; crosslinking-mass spectrometry; mitochondria; protein translocation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Chromatography, Liquid
Cross-Linking Reagents / chemistry*
HEK293 Cells
Humans
Membrane Transport Proteins / chemistry
Membrane Transport Proteins / metabolism*
Mitochondrial Membrane Transport Proteins / chemistry
Mitochondrial Membrane Transport Proteins / metabolism*
Mitochondrial Membranes / metabolism
Mitochondrial Precursor Protein Import Complex Proteins
Mitochondrial Proteins / chemistry
Mitochondrial Proteins / metabolism*
Models, Molecular
Multienzyme Complexes / chemistry*
Phosphotransferases (Alcohol Group Acceptor) / chemistry
Phosphotransferases (Alcohol Group Acceptor) / metabolism*
Protein Conformation
Protein Subunits / chemistry
Protein Subunits / metabolism
Protein Transport
Succinimides / chemistry*
Tandem Mass Spectrometry

Substances

Cross-Linking Reagents
Membrane Transport Proteins
Mitochondrial Membrane Transport Proteins
Mitochondrial Precursor Protein Import Complex Proteins
Mitochondrial Proteins
Multienzyme Complexes
Protein Subunits
Succinimides
TIM22 protein, human
TIMM29 protein, human
bis(sulfosuccinimidyl)suberate
AGK protein, human
Phosphotransferases (Alcohol Group Acceptor)