scholarly journals Mapping protein interactions in the active TOM-TIM23 supercomplex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ridhima Gomkale ◽  
Andreas Linden ◽  
Piotr Neumann ◽  
Alexander Benjamin Schendzielorz ◽  
Stefan Stoldt ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Matrix Protein ◽  
Protein Transport ◽  
Mitochondrial Proteins ◽  
Cross Linking ◽  
Intermembrane Space ◽  
Precursor Proteins ◽  
The Matrix ◽  
Targeting Signals ◽  
Chemical Cross Linking

AbstractNuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organization of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs. Here, we have designed a precursor protein that is stalled during matrix transport in a TOM-TIM23-spanning manner and enables purification of the translocation intermediate. Combining chemical cross-linking with mass spectrometric analyses and structural modeling allows us to map the molecular environment of the intermembrane space interface of TOM and TIM23 as well as the import motor interactions with amino acid resolution. Our analyses provide a framework for understanding presequence handover and translocation during matrix protein transport.

scholarly journals Coordinated Translocation of Presequence-Containing Precursor Proteins Across Two Mitochondrial Membranes: Knowns and Unknowns of How TOM and TIM23 Complexes Cooperate With Each Other

2022 ◽  
Vol 12 ◽  
Author(s):  
Marcel G. Genge ◽  
Dejana Mokranjac
Keyword(s):  
Protein Interactions ◽  
Nuclear Genome ◽  
Hydrophobic Surface ◽  
Mitochondrial Proteins ◽  
Intermembrane Space ◽  
Mitochondrial Targeting ◽  
Mitochondrial Membranes ◽  
Inner Mitochondrial Membrane ◽  
Precursor Proteins ◽  
Targeting Signals

The vast majority of mitochondrial proteins are encoded in the nuclear genome and synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals. Mitochondrial targeting signals are very diverse, however, about 70% of mitochondrial proteins carry cleavable, N-terminal extensions called presequences. These amphipathic helices with one positively charged and one hydrophobic surface target proteins to the mitochondrial matrix with the help of the TOM and TIM23 complexes in the outer and inner membranes, respectively. Translocation of proteins across the two mitochondrial membranes does not take place independently of each other. Rather, in the intermembrane space, where the two complexes meet, components of the TOM and TIM23 complexes form an intricate network of protein–protein interactions that mediates initially transfer of presequences and then of the entire precursor proteins from the outer to the inner mitochondrial membrane. In this Mini Review, we summarize our current understanding of how the TOM and TIM23 complexes cooperate with each other and highlight some of the future challenges and unresolved questions in the field.

scholarly journals Identification of Protein Transport Complexes in the Chloroplastic Envelope Membranes via Chemical Cross-Linking

1997 ◽  
Vol 136 (5) ◽  
pp. 983-994 ◽  
Author(s):  
Mitsuru Akita ◽  
Erik Nielsen ◽  
Kenneth Keegstra
Keyword(s):  
Membrane Proteins ◽  
Protein Transport ◽  
Protein Import ◽  
Cross Linking ◽  
Envelope Membrane ◽  
Precursor Proteins ◽  
Intact Chloroplasts ◽  
Outer Envelope Membrane ◽  
Envelope Membranes ◽  
Chemical Cross Linking

Transport of cytoplasmically synthesized proteins into chloroplasts uses an import machinery present in the envelope membranes. To identify the components of this machinery and to begin to examine how these components interact during transport, chemical cross-linking was performed on intact chloroplasts containing precursor proteins trapped at a particular stage of transport by ATP limitation. Large crosslinked complexes were observed using three different reversible homobifunctional cross-linkers. Three outer envelope membrane proteins (OEP86, OEP75, and OEP34) and one inner envelope membrane protein (IEP110), previously reported to be involved in protein import, were identified as components of these complexes. In addition to these membrane proteins, a stromal member of the hsp100 family, ClpC, was also present in the complexes. We propose that ClpC functions as a molecular chaperone, cooperating with other components to accomplish the transport of precursor proteins into chloroplasts. We also propose that each envelope membrane contains distinct translocation complexes and that a portion of these interact to form contact sites even in the absence of precursor proteins.

scholarly journals Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Cross-Linking Mass Spectrometry

2021 ◽  
Author(s):  
Dmitri R. Davydov ◽  
Bikash Dangi ◽  
Guihua Yue ◽  
Bhagwat Prasad ◽  
Viktor G. Zgoda
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Protein Interactions ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Cross Linking ◽  
Cytochrome P450 2E1 ◽  
Protein Protein Interactions ◽  
Chemical Cross Linking

This study aimed on exploration of the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) in the human liver on drug metabolism. Using membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide (BPM) and 4-(N-succinimidylcarboxy)benzophenone (BPS), we explored the array of its protein-protein interactions (proteome) in human liver microsomes (HLM) with chemical cross-linking mass spectrometry (CXMS). Exposure of bait-incorporated HLM samples to light was followed by isolation of the His-tagged bait protein and its cross-linked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the cross-linked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively cross-linked partners of CYP2E1 are cytochromes P450 2A6, 3A4, 2C9, and 4A11. We also detected the conjugates of CYP2E1 with UDP-glucuronosyltransferases (UGTs) 1A6, 1A9, 2B4, 2B15, and 2B17. These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes. Of particular interest is the observation of efficient cross-linking of CYP2E1 with CYP4A11. This enzyme plays a central role in the synthesis of vasoactive eicosanoids and its interactions with alcohol-inducible CYP2E1 may shed light on the mechanisms of alcohol-induced hypertension.

scholarly journals Direct observation of structure and dynamics during phase separation of an elastomeric protein

2017 ◽  
Vol 114 (22) ◽  
pp. E4408-E4415 ◽  
Author(s):  
Sean E. Reichheld ◽  
Lisa D. Muiznieks ◽  
Fred W. Keeley ◽  
Simon Sharpe
Keyword(s):  
Phase Separation ◽  
Matrix Protein ◽  
Molecular Mechanisms ◽  
Bulk Water ◽  
Extracellular Matrix Protein ◽  
Elastic Fibers ◽  
Cross Linking ◽  
Structure And Dynamics ◽  
Chemical Cross Linking

Despite its growing importance in biology and in biomaterials development, liquid–liquid phase separation of proteins remains poorly understood. In particular, the molecular mechanisms underlying simple coacervation of proteins, such as the extracellular matrix protein elastin, have not been reported. Coacervation of the elastin monomer, tropoelastin, in response to heat and salt is a critical step in the assembly of elastic fibers in vivo, preceding chemical cross-linking. Elastin-like polypeptides (ELPs) derived from the tropoelastin sequence have been shown to undergo a similar phase separation, allowing formation of biomaterials that closely mimic the material properties of native elastin. We have used NMR spectroscopy to obtain site-specific structure and dynamics of a self-assembling elastin-like polypeptide along its entire self-assembly pathway, from monomer through coacervation and into a cross-linked elastic material. Our data reveal that elastin-like hydrophobic domains are composed of transient β-turns in a highly dynamic and disordered chain, and that this disorder is retained both after phase separation and in elastic materials. Cross-linking domains are also highly disordered in monomeric and coacervated ELP3 and form stable helices only after chemical cross-linking. Detailed structural analysis combined with dynamic measurements from NMR relaxation and diffusion data provides direct evidence for an entropy-driven mechanism of simple coacervation of a protein in which transient and nonspecific intermolecular hydrophobic contacts are formed by disordered chains, whereas bulk water and salt are excluded.

scholarly journals Identification of Protein-Protein Interactions and Topologies in Living Cells with Chemical Cross-linking and Mass Spectrometry

2008 ◽  
Vol 8 (3) ◽  
pp. 409-420 ◽  
Author(s):  
Haizhen Zhang ◽  
Xiaoting Tang ◽  
Gerhard R. Munske ◽  
Nikola Tolic ◽  
Gordon A. Anderson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Living Cells ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Chemical Cross Linking

scholarly journals The unique degradation pathway of the PTS2 receptor, Pex7, is dependent on the PTS receptor/coreceptor, Pex5 and Pex20

2014 ◽  
Vol 25 (17) ◽  
pp. 2634-2643 ◽  
Author(s):  
Danielle Hagstrom ◽  
Changle Ma ◽  
Soumi Guha-Polley ◽  
Suresh Subramani
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Degradation Pathway ◽  
Matrix Proteins ◽  
Receptor Recycling ◽  
Wild Type ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signals ◽  
The Stability

Peroxisomal matrix protein import uses two peroxisomal targeting signals (PTSs). Most matrix proteins use the PTS1 pathway and its cargo receptor, Pex5. The PTS2 pathway is dependent on another receptor, Pex7, and its coreceptor, Pex20. We found that during the matrix protein import cycle, the stability and dynamics of Pex7 differ from those of Pex5 and Pex20. In Pichia pastoris, unlike Pex5 and Pex20, Pex7 is constitutively degraded in wild-type cells but is stabilized in pex mutants affecting matrix protein import. Degradation of Pex7 is more prevalent in cells grown in methanol, in which the PTS2 pathway is nonessential, in comparison with oleate, suggesting regulation of Pex7 turnover. Pex7 must shuttle into and out of peroxisomes before it is polyubiquitinated and degraded by the proteasome. The shuttling of Pex7, and consequently its degradation, is dependent on the receptor recycling pathways of Pex5 and Pex20 and relies on an interaction between Pex7 and Pex20. We also found that blocking the export of Pex20 from peroxisomes inhibits PTS1-mediated import, suggesting sharing of limited components in the export of PTS receptors/coreceptors. The shuttling and stability of Pex7 are divergent from those of Pex5 and Pex20, exemplifying a novel interdependence of the PTS1 and PTS2 pathways.

scholarly journals Compartmentation of cellular activities

2007 ◽  
Vol 28 (2) ◽  
pp. 64
Author(s):  
Trevor Lithgow
Keyword(s):  
Protein Transport ◽  
Yeast Cells ◽  
Yeast Genome ◽  
Intermembrane Space ◽  
Yeast Saccharomyces Cerevisiae ◽  
Technological Advances ◽  
The Matrix ◽  
Cellular Compartments ◽  
New Applications ◽  
Yeast Mutants

In the yeast Saccharomyces cerevisiae, almost one third of cellular function is concerned with maintaining the compartmentation of cellular activities. From classic studies in yeast genetics we have come to understand a great deal of the processes driving the delivery of proteins into these compartments and the metabolic advantages that this provides. With the publication of the yeast genome sequence, ?-omics? level studies began to provide further detail on the compartmentation of yeast cells. Very recent technological advances, including new applications in mass spectrometry, NMR, cryo-electron microscopy and the use of live-cell imaging have also been applied to yeast, because of the comparative analyses that can be done on yeast mutants. The mitochondrion is a complex compartment, carrying more than a thousand proteins that must be transported into and then distributed between, four sub-mitochondrial compartments. Essential molecular machinery in the outer and inner membranes, the intermembrane space and the matrix of mitochondria, drive protein transport, sorting and assembly. A glimpse of how S. cerevisiae and other microbes have provided understanding of cellular compartments is the aim of this review.

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