{"title":"A modified procedure for separating yeast peroxisomes from mitochondria.","authors":"Nitya Aravindan, Doron Rapaport","doi":"10.1016/bs.mie.2024.07.046","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"706 ","pages":"37-57"},"PeriodicalIF":0.0000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in enzymology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mie.2024.07.046","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/8/14 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
Abstract
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.
期刊介绍:
The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.