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Construction of a Yeast Strain for the Evaluation of Subcellular Fractionation |
HU Yan,LI Hui,HE Cheng-wen,ZHU Jing,XIE Zhi-ping() |
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China |
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Abstract Background: An eukaryotic cell relies on its cellular organelles and organelles derived compartments to perform complicated biochemical reactions efficiently. One valid way to reveal the efficient cooperation between each organelle is to isolate distinct organelles. Although several techniques are developed to separate organelles, there is almost no easy method to assess the isolation process. Goal: Construct a strain of Saccharomyces cerevisiae to evaluate the efficiency of subcellular fractionation. Methods: A detection strain of Saccharomyces cerevisiae was constructed by traditional molecular biological and cell biological methods. One soluble protein and ten membrane proteins, chosen to represent subcellular compartments as well as plasma membrane, were grouped and labeled with different epitope tags in one strain. Immunofluorescence results were compared with fluorescent protein fusions to evaluate the impact of epitope tags on the localization of these proteins. Finally, density gradient centrifugation was used to illustrate the usability of the detection strain. Results: The detetion strain labeling all major subcellular compartments of Saccharomyces cerevisiae was constructed successfully. All epitope tagged organelle markers localized to the intended subcellular sites. Each compartment of Saccharomyces cerevisiae could be detected after density gradient centrifugation. Conclusion: The detection strain is a convenient tool for the evaluation of subcellular fractionation results. It is potentially useful for future research of Saccharomyces cerevisiae cell biology.
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Received: 28 June 2020
Published: 10 November 2020
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Corresponding Authors:
Zhi-ping XIE
E-mail: zxie@sjtu.edu.cn
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|
[1] |
Wang Y, Lilley K S, Oliver S G, et al. A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation. Yeast, 2014,31(4):127-135.
doi: 10.1002/yea.3002
pmid: 24510422
|
|
|
[2] |
Stephanie E D, Scott D E, et al. Isolation of subcellular fractions from the yeast Saccharomyces cerevisiae. Current Protocols in Cell Biology, 2000,3(8):1-68.
|
|
|
[3] |
Day K J, J C Casler, B S Glick, et al. Budding yeast has a minimal endomembrane system. Dev Cell, 2018,44(1):56-72 e54.
doi: 10.1016/j.devcel.2017.12.014
pmid: 29316441
|
|
|
[4] |
Barlowe C K, Miller E A. Secretory protein biogenesis and traffic in the early secretory pathway. Genetics, 2013,193(2):383-410.
doi: 10.1534/genetics.112.142810
pmid: 23396477
|
|
|
[5] |
Gia K V, Melissa M R, Tom A R. Structural organization of the endoplasmic reticulum. EMBO Reports, 2002,3(10):944-950.
doi: 10.1093/embo-reports/kvf202
pmid: 12370207
|
|
|
[6] |
Chen S L, Novick P, Ferro-Novick S. ER structure and function. Curr Opin Cell Biol, 2013,25(4):428-433.
doi: 10.1016/j.ceb.2013.02.006
pmid: 23478217
|
|
|
[7] |
Preuss D, Mulholland J, Franzusoff A, et al. Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy. Mol Biol Cell, 1992,3:789-803.
doi: 10.1091/mbc.3.7.789
pmid: 1381247
|
|
|
[8] |
Losev E, Reinke C A, Jellen J, et al. Golgi maturation visualized in living yeast. Nature, 2006,441:1002-1006.
doi: 10.1038/nature04717
pmid: 16699524
|
|
|
[9] |
Hugh R B Pelham. Insights from yeast endosomes. Current Opinion in Cell Biology, 2002,14:454-462.
doi: 10.1016/s0955-0674(02)00352-6
pmid: 12383796
|
|
|
[10] |
Weisman L S. Yeast vacuole inheritance and dynamics. Annu Rev Gene, 2003,37:435-460.
|
|
|
[11] |
Daniel J K, Paul K H, Scott D. Emr. The fungal vacuole: composition, function, and biogenesis. Microbiological Reviews, 1990,54(3):266-292.
pmid: 2215422
|
|
|
[12] |
Joshi A S, B Nebenfuehr, V Choudhary, et al. Lipid droplet and peroxisome biogenesis occur at the same ER subdomains. Nat Commun, 2018,9(1):2940.
doi: 10.1038/s41467-018-05277-3
pmid: 30054481
|
|
|
[13] |
Wang C W. Lipid droplet dynamics in budding yeast. Cell Mol Life Sci, 2015,72(14):2677-2695.
doi: 10.1007/s00018-015-1903-5
pmid: 25894691
|
|
|
[14] |
Yuan W, M Veenhuis, I J van der Klei. The birth of yeast peroxisomes. Biochim Biophys Acta, 2016,1863(5):902-910.
pmid: 26367802
|
|
|
[15] |
Lazarow P B, Fujiki Y. Biogenesis of peroxisomes. Ann Rev Cell Bioi, 1985,1:489-530.
|
|
|
[16] |
Tabak H F, I Braakman, A vander Zand. Peroxisome formation and maintenance are dependent on the endoplasmic reticulum. Annu Rev Biochem, 2013,82:723-744.
pmid: 23414306
|
|
|
[17] |
Angela T, Susan M G. Structure and function in the budding yeast nucleus. Genetics, 2012,192:107-129.
doi: 10.1534/genetics.112.140608
pmid: 22964839
|
|
|
[18] |
Taddei A, H Schober, S M Gasser. The budding yeast nucleus. Cold Spring Harbor Perspectives in Biology, 2010,2(8):a000612.
doi: 10.1101/cshperspect.a000612
pmid: 20554704
|
|
|
[19] |
Malina C, Larsson C, Nielsen J. Yeast mitochondria: an overview of mitochondrial biology and the potential of mitochondrial systems biology. FEMS Yeast Res, 2018,18(5).
doi: 10.1093/femsyr/foy021
pmid: 29518226
|
|
|
[20] |
Xie Z P, Daniel J K. Autophagosome formation: core machinery and adaptations. Nature Cell Biology, 2007,9(10):1102-1109.
doi: 10.1038/ncb1007-1102
pmid: 17909521
|
|
|
[21] |
Aguado C, E Perez-Jimenez, M Lahuerta, et al. Isolation of lysosomes from mammalian tissues and cultured cells. Methods Mol Biol, 2016,1449:299-311.
doi: 10.1007/978-1-4939-3756-1_19
pmid: 27613045
|
|
|
[22] |
Manner A, M Islinger. Isolation of peroxisomes from rat liver and cultured hepatoma cells by density gradient centrifugation. Methods Mol Bio, 2017,1595:1-11.
|
|
|
[23] |
Huber L A, K Pfaller, I Vietor. Organelle proteomics: implications for subcellular fractionation in proteomics. Circ Res, 2003,92(9):962-968.
doi: 10.1161/01.RES.0000071748.48338.25
pmid: 12750306
|
|
|
[24] |
E Rieder, Scott D E. Overview of subcellular fractionation procedures for the yeast Saccharomyces cerevisiae. Current Protocols in Cell Biology, 2000,3(7):1-25.
|
|
|
[25] |
De Duve C. Exploring cells with a centrifuge. Nobel Lecture, 1974.
|
|
|
[26] |
Meselson M, Stahl F W, Vinograd J. Proc. Nal. Acad. Sci. U. S, 1957,43:581.
|
|
|
[27] |
Luis S M, Mercedes G De Veca, Maria Colombo, et al. Effect of pH and ATP on the equilibrium density of lysosomes. Journal of Cellular Physiology, 1993,156:303-310.
doi: 10.1002/jcp.1041560212
pmid: 8344987
|
|
|
[28] |
G Eric Schaller. Isolation of plant organelles and structures: methods and protocols. Methods in Molecular Biology, 2017,1511:119-129.
doi: 10.1007/978-1-4939-6533-5_10
pmid: 27730607
|
|
|
[29] |
Uwe Michelsen, Jörg von Hagen. Isolation of subcellular organelles and structures. Methods in Enzymology, 2009,463:305-329.
doi: 10.1016/S0076-6879(09)63019-6
pmid: 19892179
|
|
|
[30] |
Anderson N G. An introduction to particle separations in zonal centrifuges. Natl Cancer Inst Monogr, 1996,21:9-39.
pmid: 5926674
|
|
|
[31] |
Nirmal S B, R Victor Rebois. Rate zonal sedimentation of proteins in one hour or less. Analytical Biochemistry, 1997,251:103-109.
doi: 10.1006/abio.1997.2255
pmid: 9300089
|
|
|
[32] |
Wilson M A, Cascarano J. Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation. Biochem, 1972, J 129: 209-218.
|
|
|
[33] |
Earle S, Gel F. Methods in Enzymology, 1990,182:317-328.
doi: 10.1016/0076-6879(90)82027-y
pmid: 2314245
|
|
|
[34] |
Albert P K, T Page Owen, et al. Organelle isolation by magnetic immunoabsorption. BioTechniques, 1999,26(2):336-343.
doi: 10.2144/99262rr04
pmid: 10023546
|
|
|
[35] |
Takeshi N, Akira M, Yoh W, et al. Novel system for monitoring autophagy in the yeast Saccharomyces cerevisiae. Biochemical and Biophysical Research Communication, 1995,10(1):126-132.
|
|
|
[36] |
Im K, Mareninov S, Diaz M F P, et al. An introduction to performing immunofluorescence staining. Methods Mol Biol, 2019,1897:299-311.
doi: 10.1007/978-1-4939-8935-5_26
pmid: 30539454
|
|
|
[38] |
Zhu J, Zhang Z T, Tang S W, et al. A validated set of fluorescent-protein-based markers for major organelles in yeast (Saccharomyces cerevisiae). Molecular Biology and Physiology, 2019,10(5):e01691-19.
|
|
|
[39] |
Stadler C, Rexhepaj E, Singan V R, et al. Immunofluorescence and fluorescent protein tagging show high correlation for protein localization in mammalian cells. Nat Methods, 2013,10:315-323.
doi: 10.1038/nmeth.2377
pmid: 23435261
|
|
|
[40] |
Johnson M, Flick J S, Pexton T. Multiple mechanisms providerapid and stringent glucose repression of GAL gene expression in Saccharomyces cerevisiae. Mol. Cell. Biol, 1994,14:3834-3841.
doi: 10.1128/mcb.14.6.3834
pmid: 8196626
|
|
|
[41] |
Margarita C, Christian U. Purification and in vitro analysis of yeast vacuoles. Methods in Enzymology, 2008,451:177-196.
doi: 10.1016/S0076-6879(08)03213-8
pmid: 19185721
|
|
|
[42] |
Jaana M, Alex M, Paul D. Isolation of cellular lipid droplets: two purification techniques starting from yeast cells and human placentas. Journal of Visualized Experiments, 2014,86(e50981):1-10.
|
|
|
[43] |
Ben D, Astrid K. Purification of Yeast Peroxisomes, 2006,313:21-26.
|
|
|
[44] |
Istvan R B, Liza A P. Purification and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae. Methods in Cell Biology, 2007,80:45-64.
doi: 10.1016/S0091-679X(06)80002-6
pmid: 17445688
|
|
|
[45] |
Birgit S K, Rainer F, Fabienne C, et al. Partial purification and characterization of early and late endosomes from yeast. The Journal of Biological Chemistry, 1993,268(19):14376-14386.
pmid: 8314797
|
|
|
[46] |
Yu Chen Lee, Martina S G, Djuro J, et al. Plasma membrane isolation using immobilized concanavalin a magnetic beads. Liver Proteomics: Methods and Protocols, Methods in Molecular Biology, 2012,909:29-41.
doi: 10.1007/978-1-61779-959-4_3
pmid: 22903707
|
|
|
[47] |
Levental K R, Levental I. Isolation of giant plasma membrane vesicles for evaluation of plasma membrane structure and protein partitioning. Methods in Membrane Lipids, Methods in Molecular Biology, 2015,1232:65-77.
|
|
|
[48] |
Chihiro T, Yuko I A, Yuji Moriyasu. Isolation of autolysosomes from tobacco BY-2 cells. Methods in Molecular Biology, 2017,1511:151-161.
doi: 10.1007/978-1-4939-6533-5_12
pmid: 27730609
|
|
|
[49] |
Ja Yeon Kim, Wang L Y, Lee Jiyoung, et al. Hepatitis c virus induces the localization of lipid rafts to autophagosomes for its RNA replication. Journal of Virology, 2017,91(20):e00541-17.
doi: 10.1128/JVI.00541-17
pmid: 28747506
|
|
|
[50] |
G Eric Schaller. Isolation of endoplasmic reticulum and its membrane. Methods in Molecular Biology, 2017,1511:119-129.
doi: 10.1007/978-1-4939-6533-5_10
pmid: 27730607
|
|
|
[51] |
Harriet T P, Katy C, Bernhard K, et al. Isolation and proteomic characterization of the arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis. Plant Physiology, 2012,159:12-26.
pmid: 22430844
|
|
|
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