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Prediction of mitochondrial targeting signals and their cleavage sites. 线粒体靶向信号及其裂解位点的预测。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-11 DOI: 10.1016/bs.mie.2024.07.026
Fukasawa Yoshinori, Kenichiro Imai, Paul Horton
{"title":"Prediction of mitochondrial targeting signals and their cleavage sites.","authors":"Fukasawa Yoshinori, Kenichiro Imai, Paul Horton","doi":"10.1016/bs.mie.2024.07.026","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.026","url":null,"abstract":"<p><p>In this chapter we survey prediction tools and computational methods for the prediction of amino acid sequence elements which target proteins to the mitochondria. We will primarily focus on the prediction of N-terminal mitochondrial targeting signals (MTSs) and their N-terminal cleavage sites by mitochondrial peptidases. We first give practical details useful for using and installing some prediction tools. Then we describe procedures for preparing datasets of MTS containing proteins for statistical analysis or development of new prediction methods. Following that we lightly survey some of the computational techniques used by prediction tools. Finally, after discussing some caveats regarding the reliability of such methods to predict the effects of mutations on MTS function; we close with a discussion of possible future directions of computer prediction methods related to mitochondrial proteins.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"161-192"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Isolation of yeast mitochondria by differential centrifugation. 通过差速离心法分离酵母线粒体。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-03 DOI: 10.1016/bs.mie.2024.07.024
Kuo Song, Heike Rampelt
{"title":"Isolation of yeast mitochondria by differential centrifugation.","authors":"Kuo Song, Heike Rampelt","doi":"10.1016/bs.mie.2024.07.024","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.024","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"3-18"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Analysis of inner membrane lateral sorting at the presequence translocase. 前序转运酶的内膜横向分拣分析。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-21 DOI: 10.1016/bs.mie.2024.07.058
Hyun Kim
{"title":"Analysis of inner membrane lateral sorting at the presequence translocase.","authors":"Hyun Kim","doi":"10.1016/bs.mie.2024.07.058","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.058","url":null,"abstract":"<p><p>The translocase of the mitochondrial inner membrane (TIM23) complex mediates the import and membrane insertion of presequence-carrying mitochondrial proteins. It is experimentally challenging to determine whether the segment of the polypeptide is imported to the matrix or inserted into the inner membrane. Utilizing the unique topogenesis of Mgm1p, a versatile experimental approach to study the TIM23-mediated membrane insertion is developed and described in this chapter. This method combines a simple SDS-gel based assay with the quantification of the relative fractions of membrane inserted and non-inserted products, enabling the quantitative measurement of the membrane insertion efficiencies of a transmembrane segment into the mitochondrial inner membrane.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"23-38"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564284","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Analysis of quality control pathways for the translocase of the outer mitochondrial membrane. 线粒体外膜转运酶的质量控制途径分析。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-15 DOI: 10.1016/bs.mie.2024.07.050
Lara Calvo Santos, Fabian den Brave
{"title":"Analysis of quality control pathways for the translocase of the outer mitochondrial membrane.","authors":"Lara Calvo Santos, Fabian den Brave","doi":"10.1016/bs.mie.2024.07.050","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.050","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"565-584"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
STED super-resolution microscopy of mitochondrial translocases. 线粒体转运酶的 STED 超分辨率显微镜。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-21 DOI: 10.1016/bs.mie.2024.07.052
Sarah V Schweighofer, Kaushik Inamdar, Daniel C Jans, Stefan Jakobs
{"title":"STED super-resolution microscopy of mitochondrial translocases.","authors":"Sarah V Schweighofer, Kaushik Inamdar, Daniel C Jans, Stefan Jakobs","doi":"10.1016/bs.mie.2024.07.052","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.052","url":null,"abstract":"<p><p>The mitochondrial translocases of the outer membrane (TOM) and of the inner membrane (TIM) act together to facilitate the import of nuclear-encoded proteins across the mitochondrial membranes. Stimulated Emission Depletion (STED) super-resolution microscopy enables the in situ imaging of such complexes in single cells at sub-diffraction resolution. STED microscopy requires only conventional sample preparation techniques and provides super-resolved raw data without the need for further image processing. In this chapter, we provide a detailed example protocol for STED microscopy of TOM20 and mitochondrial DNA in fixed mammalian cells. The protocol includes instructions on sample preparation for immunolabeling, including cell line selection, fixation, permeabilization, blocking, labeling and mounting, but also recommendations for sample and microscope performance evaluation. The protocol is supplemented by considerations on key factors that influence the quality of the final image and also includes some considerations for the analysis of the acquired images. While the protocol described here is aimed at imaging TOM20 and DNA, it contains all the information for an immediate adaptation to other cellular targets.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"299-327"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564667","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Monitoring protein phosphorylation at the mitochondrial protein import machinery by PhosTag electrophoresis. 通过 PhosTag 电泳监测线粒体蛋白质导入机制的蛋白质磷酸化。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-09-11 DOI: 10.1016/bs.mie.2024.07.063
Fatimah Lami Imam, Chris Meisinger, Adinarayana Marada
{"title":"Monitoring protein phosphorylation at the mitochondrial protein import machinery by PhosTag electrophoresis.","authors":"Fatimah Lami Imam, Chris Meisinger, Adinarayana Marada","doi":"10.1016/bs.mie.2024.07.063","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.07.063","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"707 ","pages":"501-517"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564628","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The effect of hydrostatic pressure on lipid membrane lateral structure. 静水压力对脂膜横向结构的影响。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-16 DOI: 10.1016/bs.mie.2024.03.019
Nicola L C McCarthy, Chi L Chan, Giulia E C M Mignini Urdaneta, Yifei Liao, Robert V Law, Oscar Ces, John M Seddon, Nicholas J Brooks
{"title":"The effect of hydrostatic pressure on lipid membrane lateral structure.","authors":"Nicola L C McCarthy, Chi L Chan, Giulia E C M Mignini Urdaneta, Yifei Liao, Robert V Law, Oscar Ces, John M Seddon, Nicholas J Brooks","doi":"10.1016/bs.mie.2024.03.019","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.019","url":null,"abstract":"<p><p>High pressure is both an environmental challenge to which deep sea biology has to adapt, and a highly sensitive thermodynamic tool that can be used to trigger structural changes in biological molecules and assemblies. Lipid membranes are amongst the most pressure sensitive biological assemblies and pressure can have a large influence on their structure and properties. In this chapter, we will explore the use of high pressure small angle X-ray diffraction and high pressure microscopy to measure and quantify changes in the lateral structure of lipid membranes under both equilibrium high pressure conditions and in response to pressure jumps.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"49-76"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Using lipid binding proteins and advanced microscopy to study lipid domains. 利用脂质结合蛋白和先进的显微镜研究脂质结构域。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-04-10 DOI: 10.1016/bs.mie.2024.03.026
Nario Tomishige, Kohta Takahashi, Brigitte Pollet, Ludovic Richert, Yves Mély, Toshihide Kobayashi
{"title":"Using lipid binding proteins and advanced microscopy to study lipid domains.","authors":"Nario Tomishige, Kohta Takahashi, Brigitte Pollet, Ludovic Richert, Yves Mély, Toshihide Kobayashi","doi":"10.1016/bs.mie.2024.03.026","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.03.026","url":null,"abstract":"<p><p>Sphingomyelin is postulated to form clusters with glycosphingolipids, cholesterol and other sphingomyelin molecules in biomembranes through hydrophobic interaction and hydrogen bonds. These clusters form submicron size lipid domains. Proteins that selectively binds sphingomyelin and/or cholesterol are useful to visualize the lipid domains. Due to their small size, visualization of lipid domains requires advanced microscopy techniques in addition to lipid binding proteins. This Chapter describes the method to characterize plasma membrane sphingomyelin-rich and cholesterol-rich lipid domains by quantitative microscopy. This Chapter also compares different permeabilization methods to visualize intracellular lipid domains.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"700 ","pages":"217-234"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141545062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Quantifying uncertainty in trans-membrane stresses and moments in simulation. 在模拟中量化跨膜应力和力矩的不确定性。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-05-17 DOI: 10.1016/bs.mie.2024.04.008
Samuel L Foley, Markus Deserno
{"title":"Quantifying uncertainty in trans-membrane stresses and moments in simulation.","authors":"Samuel L Foley, Markus Deserno","doi":"10.1016/bs.mie.2024.04.008","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.04.008","url":null,"abstract":"<p><p>The lateral stress profile of a lipid bilayer constitutes a valuable link between molecular simulation and mesoscopic elastic theory. Even though it is frequently calculated in simulations, its statistical precision (or that of observables derived from it) is often left unspecified. This omission can be problematic, as uncertainties are prerequisite to assessing statistical significance. In this chapter, we provide a comprehensive yet accessible overview of the statistical error analysis for the lateral stress profile. We detail two relatively simple but powerful techniques for generating error bars: block-averaging and bootstrapping. Combining these methods allows us to reliably estimate uncertainties, even in the presence of both temporal and spatial correlations, which are ubiquitous in simulation data. We illustrate these techniques with simple examples like stress moments, but also more complex observables such as the location of stress profile extrema and the monolayer neutral surface.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"701 ","pages":"83-122"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141723931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Determining the esterase activity of peptides and peptide assemblies. 确定肽和肽组合物的酯酶活性。
4区 生物学
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-02-20 DOI: 10.1016/bs.mie.2024.02.002
Patrizia Janković, Daniela Kalafatovic
{"title":"Determining the esterase activity of peptides and peptide assemblies.","authors":"Patrizia Janković, Daniela Kalafatovic","doi":"10.1016/bs.mie.2024.02.002","DOIUrl":"10.1016/bs.mie.2024.02.002","url":null,"abstract":"<p><p>Catalytic peptides are gaining attention as alternatives to enzymes, especially in industrial applications. Recent advances in peptide design have improved their catalytic efficiency with approaches such as self-assembly and metal ion complexation. However, the fundamental principles governing peptide catalysis at the sequence level are still being explored. Ester hydrolysis, a well-studied reaction, serves as a widely employed method to evaluate the catalytic potential of peptides. The standard colorimetric reaction involving para-nitrophenyl acetate hydrolysis acts as a benchmark assay, providing a straightforward and efficient screening method for rapidly identifying potential catalysts. However, maintaining standardized conditions is crucial for reproducible results, given that factors such as pH, temperature, and substrate concentration can introduce unwanted variability. This necessity becomes particularly pronounced when working with peptides, which often exhibit slower reaction rates compared to enzymes, making even minor variations significantly influential on the final outcome. In this context, we present a refined protocol for assessing the catalytic activity of peptides and peptide assemblies, addressing critical considerations for reproducibility and accuracy.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"697 ","pages":"423-433"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141180207","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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