bims-mitpro 2024-11-10 papers

Issue of 2024–11–10
five papers selected by
Andreas Kohler, Umeå University

Methods Enzymol. 2024 ;pii: S0076-6879(24)00395-1. [Epub ahead of print]707 565-584

Analysis of quality control pathways for the translocase of the outer mitochondrial membrane.

The functionality of mitochondria depends on the import of proteins synthesized on cytosolic ribosomes. Impaired import into mitochondria results in mitochondrial dysfunction and proteotoxic accumulation of precursor proteins in the cytosol. All proteins sorted to inner mitochondrial compartments are imported via the translocase of the outer membrane (TOM) complex. Premature protein folding, a reduction of the mitochondrial membrane potential or defects in translocases can result in precursor arrest during translocation, thereby clogging the TOM channel and blocking protein import. In recent years, different pathways have been identified, which employ the cytosolic ubiquitin-proteasome system in the extraction and turnover of precursor proteins from the TOM complex. Central events in this process are the modification of arrested precursor proteins with ubiquitin, their extraction by AAA-ATPases and subsequent degradation by the 26 S proteasome. Analysis of these processes is largely facilitated by the expression of model proteins that function as efficient "cloggers" of the import machinery. Here we describe the use of such clogger proteins and how their handling by the protein quality control machinery can be monitored. We provide protocols to study the extent of clogging, the ubiquitin-modification of arrested precursor proteins and their turnover by the 26 S proteasome.

Keywords: Mitochondria; Proteasome; Protein import; Protein quality control; Ubiquitin

DOI: https://doi.org/10.1016/bs.mie.2024.07.050

Methods Enzymol. 2024 ;pii: S0076-6879(24)00408-7. [Epub ahead of print]707 501-517

Monitoring protein phosphorylation at the mitochondrial protein import machinery by PhosTag electrophoresis.

Fatimah Lami Imam, Chris Meisinger, Adinarayana Marada.

The mitochondrial import machinery is regulated by several protein kinases that phosphorylate key components. This allows an adjustment of the protein flux to changing cellular demands and allow a dynamic organellar proteome. PhosTag electrophoresis has been proven as highly valuably tool to study these signalling machanisms at the import machinery.

Keywords: TOM complex; assembly; biogenesis; import machinery; precursor protein; protein kinase; reversible phosphorylation; signalling

DOI: https://doi.org/10.1016/bs.mie.2024.07.063

Methods Enzymol. 2024 ;pii: S0076-6879(24)00365-3. [Epub ahead of print]707 543-564

Methods to analyze the mitochondrial unfolded protein response (UPRmt).

Avijit Mallick, Cole M Haynes.

The mitochondrial unfolded protein response (UPRmt) is a mitochondria-to-nuclear signaling pathway that mediates the transcription of genes required to maintain mitochondrial function during development as well as during aging. In this chapter, we describe the approaches and techniques that we and others have used to elucidate the mechanism(s) by which cells detect mitochondrial stress or dysfunction and communicate with the nucleus to induce transcription of a protective stress response. We also describe approaches to evaluate the impact of UPRmt activation on mitochondrial function and mitochondrial biogenesis including imaging-based approaches as well as approaches to evaluate mitochondrial genome (mtDNA) copy number.

Keywords: Deleterious mtDNA heteroplasmy; Mito-nuclear communication; Mitochondrial biogenesis; Mitochondrial unfolded protein response; Molecular chaperones; MtDNA replication

DOI: https://doi.org/10.1016/bs.mie.2024.07.029

Methods Enzymol. 2024 ;pii: S0076-6879(24)00400-2. [Epub ahead of print]707 39-62

Monitoring α-helical membrane protein insertion into the outer mitochondrial membrane of yeast cells.

Isabelle Steymans, Thomas Becker.

Mitochondria are surrounded by two membranes, the outer and inner membrane. The outer membrane contains a few dozen integral membrane proteins that mediate transport, fusion and fission processes, form contact sites and are involved in signaling pathways. There are two different types of outer membrane proteins. A few proteins are membrane-integrated by a transmembrane β-barrel, while other proteins are embedded by single or multiple α-helical membrane segments. All outer membrane proteins are produced on cytosolic ribosomes, but their import mechanisms differ. The translocase of the outer mitochondrial membrane (TOM complex) and the sorting and assembly machinery (SAM complex) import β-barrel proteins. Different import pathways have been reported for proteins with α-helical membrane anchors. The mitochondrial import (MIM) complex is the major insertase for this type of proteins. The in vitro import of radiolabeled precursor proteins into isolated mitochondria is a versatile technique to study protein import into the outer mitochondrial membrane. The import of these proteins does not involve proteolytic processing and is not dependent on the membrane potential. Therefore, the import assay has to be combined with blue native electrophoresis, carbonate extraction or protease accessibility assays to determine the import efficiency. These techniques allow to define import steps, assembly intermediates and study membrane integration. The in vitro import assay has been a central tool to uncover specific import routes and mechanisms.

Keywords: MIM complex; Mitochondria; Protein sorting; SAM complex; TOM complex

DOI: https://doi.org/10.1016/bs.mie.2024.07.055

Methods Enzymol. 2024 ;pii: S0076-6879(24)00425-7. [Epub ahead of print]707 585-610

Analysis of the mitochondria-associated degradation pathway (MAD) in yeast cells.

Pin-Chao Liao, Liza A Pon.

Mitochondria are critical for cellular function in health, disease and aging. Mitochondria-associated degradation (MAD), a pathway for quality control of the organelle, recognizes and ubiquitinates unfolded mitochondrial proteins, removes them from the organelle using a conserved segregase complex, which contains an AAA-ATPase Cdc48 and its cofactors, and degrades them using the ubiquitin-proteasome system (UPS). Here, we describe an approach to (1) study the turnover and ubiquitination of candidate MAD substrates, (2) assay retrotranslocation and export of MAD substrates from the mitochondrial matrix in vitro, and (3) study interactions between MAD substrates and Cdc48 using the budding yeast, Saccharomyces cerevisiae, as a model organism.

Keywords: Affinity purification; Budding yeast; Co-immunoprecipitation; Mitochondrial isolation; Mitochondrial quality control

DOI: https://doi.org/10.1016/bs.mie.2024.09.001