bims-tricox 2024-11-03 papers

bims-tricox

Biomed News

on Translation, ribosomes and COX

Issue of 2024–11–03
twelve papers selected by
Yash Verma, University of Zurich

In vitro import of mitochondrial precursor proteins into yeast mitochondria.
Assays to monitor mitochondrial translation.
Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol.
Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.
Monitoring mitochondrial protein import by live cell imaging.
In organello silencing of mitochondrial gene expression.
Import of mitochondrial precursor proteins into mitochondria of semi-intact yeast cells.
Analysis and prediction of internal mitochondrial targeting signals.
A modified procedure for separating yeast peroxisomes from mitochondria.
Isolation of yeast mitochondria by differential centrifugation.
Subcellular fractionation by differential centrifugation for mitochondrial studies.
Analysis of mitochondrial biogenesis and protein localization by genetic screens and automated imaging.

Methods Enzymol. 2024 ;pii: S0076-6879(24)00342-2. [Epub ahead of print]706 347-363

In vitro import of mitochondrial precursor proteins into yeast mitochondria.

Soraya Badrie, Julian Alexander Draken, Dejana Mokranjac.

  Mitochondria contain about 1000 different proteins, only a handful of which are encoded in the mitochondrial genome. The remaining c. 99% of mitochondrial proteins are encoded in the nuclear genome, synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals and are subsequently imported into the organelle. Mitochondrial targeting signals are very diverse and mitochondria therefore also have a number of very sophisticated molecular machines that recognize, import and sort mitochondrial precursor proteins to the different mitochondrial subcompartments. The ability to synthesize mitochondrial precursor proteins in vitro and subsequently import them into isolated mitochondria has revolutionized our understanding of mitochondrial protein import pathways. Here, we describe the basic protocol for synthesis of mitochondrial precursor proteins in vitro and their subsequent import into isolated mitochondria from yeast Saccharomyces cerevisiae, the method which was used to elucidate and characterize the vast majority of mitochondrial protein import pathways.

Keywords:  (35)S-methionine; In vitro import; In vitro transcription and translation; Isolated mitochondria; Protein translocation; Saccharomyces cerevisiae

DOI:  https://doi.org/10.1016/bs.mie.2024.07.016
Methods Enzymol. 2024 ;pii: S0076-6879(24)00385-9. [Epub ahead of print]706 519-532

Assays to monitor mitochondrial translation.

Sung-Jun Jung, Martin Ott.

  The complexes of the oxidative phosphorylation (OXPHOS) system found in the mitochondrial inner membrane comprises nuclear and mitochondrial-encoded proteins. The mitochondrial-encoded subunits of the OXPHOS complexes play vital catalytic roles for OXPHOS. These subunits are inserted co-translationally into the inner membrane, where they are matured and assembled with nuclear encoded subunits, requiring a set of OXPHOS assembly and quality control factors. Hence, monitoring the fate of newly synthesized mitochondrial-encoded polypeptides is a basic and essential approach for exploring OXPHOS biogenesis and the related protein quality control processes. Here, we describe a detailed protocol for labeling mitochondrial encoded proteins with 35S-methionine for pulse and pulse/chase experiments, both in vivo and in organello, using the yeast Saccharomyces cerevisiae as the model. These methods enable analyses of the early steps during the biogenesis and turnover of mitochondrial-encoded proteins.

Keywords:  35S-methionine; Mitochondrial translation; isolated mitochondria; protein stability; protein synthesis; yeast

DOI:  https://doi.org/10.1016/bs.mie.2024.07.045
Methods Enzymol. 2024 ;pii: S0076-6879(24)00356-2. [Epub ahead of print]706 287-311

Monitoring and analysis of mitochondrial precursor protein aggregates in the cytosol.

Klaudia K Maruszczak, Agnieszka Chacinska.

  The vast majority of mitochondrial precursor proteins is synthesized in the cytosol and subsequently imported into the organelle with the help of targeting signals that are present within these proteins. Disruptions in mitochondrial import will result in the accumulation of the organellar precursors in the cytosol of the cell. If mislocalized proteins exceed their critical concentrations, they become prone to aggregation. Under certain circumstances, protein aggregation becomes an irreversible process, which eventually endangers cellular health. Impairment in mitochondrial biogenesis and its effect on cellular protein homeostasis were recently linked to neurodegeneration, therefore placing this process in the center of attention. In this chapter, we are presenting a set of techniques that allows to monitor and study mitochondrial precursor protein aggregates upon mitochondrial dysfunction in the cytosol of both yeast and human cells.

Keywords:  Mitochondria; Mitochondrial dysfunction; Mitochondrial import; Protein aggregates

DOI:  https://doi.org/10.1016/bs.mie.2024.07.020
Methods Enzymol. 2024 ;pii: S0076-6879(24)00370-7. [Epub ahead of print]706 365-390

Monitoring the in vitro import and assembly of mitochondrial precursor proteins into mammalian mitochondria.

Jordan J Crameri, Diana Stojanovski.

  Mitochondrial protein import is a complex process governing the delivery of the organelle's proteome. This process, in turn, is essential for maintaining mitochondrial function and cellular homeostasis. Initiated by protein synthesis in the cytoplasm, precursor proteins destined for the mitochondria possess targeting signals that guide them to the mitochondrial surface. At mitochondria, the translocation of proteins across the mitochondrial membranes involves an intricate interplay between translocases, chaperones, and receptors. The mitochondrial import assay offers researchers the opportunity to recapitulate the process of protein import in vitro. The assay has served as an indispensable tool in helping decipher the intricacies of protein translocation into mitochondria, first in fungal models, and subsequently in higher eukaryotic models. In this chapter, we will describe how protein import can be assayed using mammalian mitochondria and provide insight into the types of questions that can be addressed in mammalian mitochondrial biology using this experimental approach.

Keywords:  in vitro; mitochondria; protein import; translocase

DOI:  https://doi.org/10.1016/bs.mie.2024.07.034
Methods Enzymol. 2024 ;pii: S0076-6879(24)00363-X. [Epub ahead of print]706 437-447

Monitoring mitochondrial protein import by live cell imaging.

F X Reymond Sutandy.

  The majority of mitochondrial proteins are synthesized in the cytosol and must be imported into mitochondria to attain their mature forms and execute their functions. Disruption of mitochondrial functions, whether caused by external or internal stress, may compromise mitochondrial protein import. Therefore, monitoring mitochondrial protein import has become a standard approach to assess mitochondrial health and gain insights into mitochondrial biology, especially during stress. This chapter describes a detailed protocol for monitoring mitochondrial import in live cells using microscopy. Co-localization between mitochondria and a genetic reporter of mitochondrially targeted enhanced GFP (eGFP) is employed to evaluate mitochondrial protein import efficiency under different physiological conditions. Overall, this technique provides a simple and robust approach to assess mitochondrial protein import efficiency within its native cellular environment.

Keywords:  MTS; mitochondria; protein import; stress response

DOI:  https://doi.org/10.1016/bs.mie.2024.07.027
Methods Enzymol. 2024 ;pii: S0076-6879(24)00371-9. [Epub ahead of print]706 501-518

In organello silencing of mitochondrial gene expression.

Mats Koschel, Luis Daniel Cruz-Zaragoza.

  Mitochondria contain proteins from two genetic origins. Most mitochondrial proteins are encoded in the nuclear genome, translated in the cytosol, and subsequently imported into the different mitochondrial sub-compartments. A small number is encoded in the mitochondrial DNA (mtDNA). The manipulation of the mtDNA gene expression represents a challenge. Here, we present an in vitro approach using morpholinos chemically linked to a precursor protein to silence gene expression in purified human mitochondria. The protocol is demonstrated with a Jac1-morpholino chimera specifically targeting COX1 mRNA. The chimera import and mitochondrial translation requirements are described in a step-by-step procedure, where the dose-dependent effect of reducing COX1 translation is observed. The affinity and specificity of chimera-mRNA binding also show great applicability to purify transcript-associated proteins by using the imported chimera construct as bait for immunoprecipitation. This new strategy opens up the possibility to address mechanistic questions about gene expression and physiology in mitochondria.

Keywords:  Gene expression; In vitro; Mitochondria; Morpholino; Silencing

DOI:  https://doi.org/10.1016/bs.mie.2024.07.035
Methods Enzymol. 2024 ;pii: S0076-6879(24)00359-8. [Epub ahead of print]706 391-405

Import of mitochondrial precursor proteins into mitochondria of semi-intact yeast cells.

Johannes M Herrmann, Svenja Lenhard, Katja G Hansen.

  Mitochondria import hundreds of different precursor proteins from the cytosol and direct each of these to its specific mitochondrial subcompartment. The import routes and mechanisms by which precursors are transported into the outer membrane, the intermembrane space (IMS), the inner membrane and the matrix have been characterized in depth by use of very powerful in vitro assays. In the 'classical' import assays, radiolabeled precursor proteins are incubated with isolated mitochondria and the protein uptake is monitored by one or more of the following observations: intramitochondrial processing, resistance to externally added proteases, or the formation of disulfide bonds. In this chapter, we describe an alternative import assay which employs semi-intact yeast cells. This assay uses spheroplasts from which the cell wall had been removed by enzymatic digestion before the plasma membrane was partially permeabilized by a freeze-thawing step. Since the organellar architecture is largely maintained in semi-intact cells, this in vitro import assay allows to elucidate the targeting of precursor proteins from the cytoplasm to the mitochondrial surface. Thereby the contribution of other compartments such as the endoplasmic reticulum (ER) can be assessed. Here we describe how semi-intact cells are prepared and used in the in vitro import assay and discuss the pros and cons of this approach.

Keywords:  Intracellular targeting; Mitochondria; Organellar contact sites; Protein import; Protein targeting; Radiolabeled precursor proteins; Spheroplasts

DOI:  https://doi.org/10.1016/bs.mie.2024.07.023
Methods Enzymol. 2024 ;pii: S0076-6879(24)00378-1. [Epub ahead of print]706 263-283

Analysis and prediction of internal mitochondrial targeting signals.

Felix Jung, Saskia Rödl, Johannes M Herrmann, Timo Mühlhaus.

  Mitochondria consist of several hundreds of proteins, the vast majority of which are synthesized in the cytosol as precursor proteins from where they are targeted to and imported into mitochondria. The transport of proteins into mitochondria relies on specific targeting information encoded within the protein sequence, known as mitochondrial targeting sequences (MTSs). These N-terminal extensions are usually between 8 and 80 residues long and form amphipathic helices with one hydrophobic and one positively charged surface. Receptors on the mitochondrial surface recognize the MTSs and direct precursors through protein-conducting channels in the outer and inner membrane to the mitochondrial matrix, where presequences are often removed by proteases. In addition to these MTSs, many mitochondrial proteins contain internal matrix targeting sequences (iMTSs) which share the same structural features with MTSs. These iMTSs are neither necessary nor sufficient for mitochondrial targeting, however, they help to increase the import-competence of precursor proteins as they bind to the TOM receptors and presumably facilitate the unfolding of precursors on the mitochondrial surface. Prediction algorithms allow the identification of iMTSs in protein sequences. In this chapter, we present iMLP, an agnostic algorithm for the prediction of iMTS propensity profiles. This iMTS prediction tool is provided via an iMLP webservice at http://iMLP.bio.uni-kl.de and is also available as a BioFSharp application that can be executed locally. We describe and explain the usage of this prediction algorithm and how to interpret the results of this valuable tool.

Keywords:  IMTS; Machine learning; Mitochondria; Presequence; Protein targeting; Sequence analysis; TargetP; Targeting prediction; Tom70; Webservice

DOI:  https://doi.org/10.1016/bs.mie.2024.07.038
Methods Enzymol. 2024 ;pii: S0076-6879(24)00386-0. [Epub ahead of print]706 37-57

A modified procedure for separating yeast peroxisomes from mitochondria.

Nitya Aravindan, Doron Rapaport.

  Mitochondria and peroxisomes are mutually dependent organelles that share several membrane proteins that carry out the same function in both organelles. To study the unique features of these dually localized proteins in each of the two organelles, it is essential to separate mitochondria from peroxisomes. Isolating organelles from cells of Baker's yeast, Saccharomyces cerevisiae, is crucial for our understanding of the biogenesis and functions of proteins. Traditionally, subcellular fractionation and isolation of individual organelles by differential centrifugation benefit from the specific and unique density of each organelle. However, when yeast cells are grown under normal conditions, certain organelles like mitochondria and peroxisomes share strikingly similar densities. This similarity challenges the separation of these organelles from one another. In this chapter, we describe an optimized procedure to address this task. We depict growth conditions that would favor stimulation of peroxisomes to increase their number and density, and portray organellar isolation followed by gradient centrifugation, enabling an improved separation of both organelles. Additionally, we illustrate the advantage of the procedure to study the dual localization of the membrane protein Fis1.

Keywords:  Density gradient; Fis1; Mitochondria; Peroxisomes; Subcellular fractionation; Yeast

DOI:  https://doi.org/10.1016/bs.mie.2024.07.046
Methods Enzymol. 2024 ;pii: S0076-6879(24)00360-4. [Epub ahead of print]706 3-18

Isolation of yeast mitochondria by differential centrifugation.

Kuo Song, Heike Rampelt.

  The isolation of intact and functional mitochondria is a powerful approach to characterize and study this organelle. The classical biochemical method of differential centrifugation is routinely used to isolate mitochondria. This method has several advantages, such as a high yield and easy adaptability. The isolated mitochondria are physiologically active and can be used for a variety of follow-up experiments, for example protein import and respiration measurements. Here, we describe the procedure to purify mitochondria from the budding yeast Saccharomyces cerevisiae. In addition, two approaches are introduced to assess the quality of isolated mitochondria, by limited proteinase K digestion or measurement of the membrane potential.

Keywords:  Mitochondrial preparation; fractionation; limited proteolysis; membrane potential; organelle isolation

DOI:  https://doi.org/10.1016/bs.mie.2024.07.024
Methods Enzymol. 2024 ;pii: S0076-6879(24)00377-X. [Epub ahead of print]706 61-73

Subcellular fractionation by differential centrifugation for mitochondrial studies.

Conny Steiert, Jon V Busto, Laura Melchionda, Nils Wiedemann.

  In addition to fluorescence microscopy, the subcellular fractionation of eukaryotic cells remains one of the central methods for the basic characterization of proteins. Here we describe an optimized procedure for the subcellular fractionation of yeast cells, specifically for mitochondrial studies. Major recommendations are to separate the fractions immediately after each centrifugation step, to carefully discard a significant part of the supernatant fractions which is in the direct vicinity to the pellets and, in addition, to perform an extra homogenization step of the post nuclear supernatant fraction. These principles help to collect supernatant fractions with less cross-contaminations from the corresponding pellets. These approaches are scalable and adaptable for the fractionation of other cell types and are also useful for the characterization of other organelles.

Keywords:  Cell organelles; Cytosol; Endoplasmic reticulum; Microsomes; Mitochondria; Nucleus; Post nuclear supernatant; Saccharomyces cerevisiae; Yeast

DOI:  https://doi.org/10.1016/bs.mie.2024.07.037
Methods Enzymol. 2024 ;pii: S0076-6879(24)00358-6. [Epub ahead of print]706 97-123

Analysis of mitochondrial biogenesis and protein localization by genetic screens and automated imaging.

Yury S Bykov, Maya Schuldiner.

  Budding yeast is a laboratory model of a simple eukaryotic cell. Its compact genome is very easy to edit. This allowed to create systematic collections (libraries) of yeast strains where every gene is either perturbed or tagged. Here we review how such collections were used to study mitochondrial biology by doing genetic screens. First, we introduce the principles of yeast genome editing and the basics of its life cycle that are useful for genetic experiments. Then we overview what yeast strain collections were created over the past years. We also describe the creation and the usage of the new generation of SWAP-Tag (SWAT) collections that allow to create custom libraries. We outline the principles of changing the genetic background of whole collections in parallel, and the basics of synthetic genetic array (SGA) approach. Then we review the discoveries that were made using different types of genetic screens focusing on general mitochondrial functions, proteome, and protein targeting pathways. The development of new collections and screening techniques will continue to bring valuable insight into the function of mitochondria and other organelles.

Keywords:  High-throughput genetics; Mitochondria; Protein targeting; Proteome; Swap-Tag (SWAT); Synthetic genetic array; Yeast; Yeast collections

DOI:  https://doi.org/10.1016/bs.mie.2024.07.022