bims-mitpro 2024-11-03 papers

bims-mitpro

Biomed News

on Mitochondrial proteostasis

Issue of 2024–11–03
five papers selected by
Andreas Kohler, Umeå University

The Integrated Stress Response Suppresses PINK1-dependent Mitophagy by Preserving Mitochondrial Import Efficiency.
Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.
Analysis of targeting signals for mitochondrial intermembrane space import.
Quantifying mitochondrial protein import by mePRODmt proteomics.
The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.

bioRxiv. 2024 Oct 17. pii: 2024.10.16.617214. [Epub ahead of print]

The Integrated Stress Response Suppresses PINK1-dependent Mitophagy by Preserving Mitochondrial Import Efficiency.

Mingchong Yang, Zengshuo Mo, Kelly Walsh, Wen Liu, Xiaoyan Guo.

Mitophagy is crucial for maintaining mitochondrial health, but how its levels adjust to different stress conditions remains unclear. In this study, we investigated the role of the DELE1-HRI axis of integrated stress response (ISR) in regulating mitophagy, a key mitochondrial stress pathway. Our findings show that the ISR suppresses mitophagy under non-depolarizing mitochondrial stress by positively regulating mitochondrial protein import, independent of ATF4 activation. Mitochondrial protein import is regulated by the rate of protein synthesis under both depolarizing and non-depolarizing stress. Without ISR, increased protein synthesis overwhelms the mitochondrial import machinery, reducing its efficiency. Under depolarizing stress, mitochondrial import is heavily impaired even with active ISR, leading to significant PINK1 accumulation. In contrast, non-depolarizing stress allows more efficient protein import in the presence of ISR, resulting in lower mitophagy. Without ISR, mitochondrial protein import becomes severely compromised, causing PINK1 accumulation to reach the threshold necessary to trigger mitophagy. These findings reveal a novel link between ISR-regulated protein synthesis, mitochondrial import, and mitophagy, offering potential therapeutic targets for diseases associated with mitochondrial dysfunction.

DOI: https://doi.org/10.1101/2024.10.16.617214
J Cell Sci. 2024 Oct 28. pii: jcs.263548. [Epub ahead of print]

Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.

Marta Lopez-Nieto, Zhaozhi Sun, Emily Relton, Rahme Safakli, Brian D Freibaum, J Paul Taylor, Alessia Ruggieri, Ioannis Smyrnias, Nicolas Locker.

  To rapidly adapt to harmful changes to their environment, cells activate the integrated stress response (ISR). This results in an adaptive transcriptional and translational rewiring, and the formation of biomolecular condensates named stress granules (SGs), to resolve stress. In addition to this first line of defence, the mitochondrial unfolded protein response (UPRmt) activates a specific transcriptional programme to maintain mitochondrial homeostasis. We present evidence that SGs and UPRmt pathways are intertwined and communicate. UPRmt induction results in eIF2a phosphorylation and the initial and transient formation of SGs, which subsequently disassemble. The induction of GADD34 during late UPRmt protects cells from prolonged stress by impairing further assembly of SGs. Furthermore, mitochondrial functions and cellular survival are enhanced during UPRmt activation when SGs are absent, suggesting that UPRmt-induced SGs have an adverse effect on mitochondrial homeostasis. These findings point to a novel crosstalk between SGs and the UPRmt that may contribute to restoring mitochondrial functions under stressful conditions.

Keywords:  GADD34; Integrated stress response; Mitochondrial stress response; Stress granules; UPRmt

DOI:  https://doi.org/10.1242/jcs.263548
Methods Enzymol. 2024 ;pii: S0076-6879(24)00366-5. [Epub ahead of print]706 243-262

Analysis of targeting signals for mitochondrial intermembrane space import.

Kostas Tokatlidis, Amiyo Haider.

  The mitochondrial intermembrane space (IMS) is the smallest sub-mitochondrial compartment, containing only 5%-10% of mitochondrial proteins. Despite its size, it exhibits the most diverse array of protein import mechanisms. These are underpinned by several different types of targeting signals that are quite distinct from targeting signals for other mitochondrial sub-compartments. In this chapter we outlined our current understanding of some of the main IMS import pathways, the primary oxidative protein folding targeting signal, and explore the remarkable variety of alternative import methods. Unlike proteins destined for the matrix or inner membrane (IM), IMS proteins need only traverse the outer mitochondrial membrane. This process doesn't require energy from ATP hydrolysis in the matrix or the IM electrochemical potential. We also examine unconventional IMS import pathways that remain poorly understood, often guided by ill-defined or unknown targeting peptides. Many IMS proteins are implicated in human diseases, making it crucial to comprehend how they reach their functional location within the IMS. The chapter concludes by discussing current insights into how understanding IMS targeting pathways can contribute to improved understanding of a wide range of human disorders.

Keywords:  Chaperones; Disulfide bonds; In vitro protein import; Intermembrane space; MIA pathway; Oxidative folding; Redox; Targeting

DOI:  https://doi.org/10.1016/bs.mie.2024.07.030
Methods Enzymol. 2024 ;pii: S0076-6879(24)00347-1. [Epub ahead of print]706 449-474

Quantifying mitochondrial protein import by mePRODmt proteomics.

Süleyman Bozkurt, Bhavesh S Parmar, Christian Münch.

  Mitochondrial protein import is crucial for maintaining cellular health and homeostasis. Disruptions in this process have been linked to various diseases. Traditional methods for studying mitochondrial protein import predominantly focus on individual proteins and lack the dynamic resolution needed to fully appreciate the complexity of mitochondrial proteostasis and protein trafficking. To address these limitations, we developed a technique called mitochondria-specific multiplexed enhanced protein dynamics (mePRODmt). This method is a novel application of the mePROD methodology and utilizes pulsed stable isotope labeling with amino acids in cell culture (pSILAC)-based proteomics approach to study transient mitochondrial protein import. This chapter outlines the mePRODmt protocol, which includes the preparation of heavy SILAC-labeled peptides for boosting overall mitochondrial peptide signals (booster), SILAC labeling of cultured cells under experimental conditions, mitochondria isolation, sample preparation for multiplex proteomics using tandem mass tags (TMT) for isobaric labeling, recommended liquid chromatography-mass spectrometry (LC-MS) settings for reporter ion quantitation and a data analysis pipeline to analyze pSILAC-TMT data.

Keywords:  Mass spectrometry; Mitochondria; Mitochondrial protein import; Proteomics; SILAC; TMT multiplex; Translation; mePROD; pSILAC

DOI:  https://doi.org/10.1016/bs.mie.2024.07.017
Methods Enzymol. 2024 ;pii: S0076-6879(24)00369-0. [Epub ahead of print]706 407-436

The MitoLuc assay for the analysis of the mechanism of mitochondrial protein import.

Hope I Needs, Youmian Yan, Natalie M Niemi, Ian Collinson.

  The NanoLuc split luciferase assay has proven to be a powerful tool for the analysis of protein translocation. Its flexibility has enabled in vivo, ex vivo, and in vitro studies-including systems reconstituting protein transport from pure components. The assay has been particularly useful in the characterization of bacterial secretion and mitochondrial protein import. In the latter case, MitoLuc has been developed for the investigation of the TIM23-pathway via import into the matrix of isolated yeast mitochondria. Subsequent analysis identified three distinct phases of import, rather than in a single continuous step. The assay has also been developed to monitor import into the mitochondrial matrix of intact cultured cells. This latter innovation has laid the foundations for further analysis of the import process in humans, including the consequences of interactions with cytosolic factors and neighboring organelles. The versatility of the MitoLuc assay is conducive for its adaptation to also monitor import into the inter-membrane space (MIA-pathway), and into the inner-membrane via the TIM22- and TIM23-complexes. Here, we present detailed protocols for the application of MitoLuc to mitochondria isolated from yeast and to those within cultured human cells.

Keywords:  Cell culture; Luciferase; MitoLuc assay; Mitochondrial biogenesis; Mitochondrial protein import; NanoLuc; Protein translocation; Yeast

DOI:  https://doi.org/10.1016/bs.mie.2024.07.033