Construction of a Yeast Strain for the Evaluation of Subcellular Fractionation

doi:10.13523/j.cb.2006053

China Biotechnology

2020, Vol. 40

Issue (10): 10-23 DOI: 10.13523/j.cb.2006053

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.

Key words： Yeast Organelles Autophagosome Plasma membrane Subcellular fractionation

Received: 28 June 2020 Published: 10 November 2020

ZTFLH:

Q819

Corresponding Authors: Zhi-ping XIE E-mail: zxie@sjtu.edu.cn

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Cite this article:

HU Yan,LI Hui,HE Cheng-wen,ZHU Jing,XIE Zhi-ping. Construction of a Yeast Strain for the Evaluation of Subcellular Fractionation. China Biotechnology, 2020, 40(10): 10-23.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2006053 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I10/10

Table 1 Marker protein of subcellular conpartments in Saccharomyces cerevisiae

Table 2 Plasmids and their restriction sites

Table 3 Large plasmids and their restriction sites

Table 4 Yeast strain list

Fig.1 Western blot results of YOT027 (a)Flag tag(Vph1, Nab2, Erg6) (b) Myc tag(Emc1, Pex3, Cox4) (c) V5 tag(Chs5, Anp1, Snf7) (d) Vsv tag(Pma1, Atg8)

Fig.2 Conlocalization between immunofluorescence and fluorescence proteins (a) Emc1-4myc and Emc1-mCherry (b) Vph1-2flag and Vph1-mCherry (c) Cox4-12myc and Cox4-DuDre (d) Pma1-3vsv and Pma1-GFP (e) Nab2-2flag, Nab2-mCherry and Nab2(DAPI) (f) Pex3-4myc and Pex3-DuDre (g) Erg6-2flag and Erg6-mCherry (h) Anp1-4v5 and Anp1-mCherry (i) Chs5-4v5 and Chs5-mCherry (j) Snf7-4v5 and Snf7-mCherry (k) 9vsv-Atg8 and GFP-Atg8

Fig.3 Test of different immunofluorescence conditions Test of different perforation buffer of immunofluorescence by detecting the colocalization between Pma1-3vsv and Pma1-GFP. The best one is PBS, 1mg/mL BSA, 0.2% Triton×100, 0.5% SDS and 2.4mol/L sorbitol

Fig.4 Density gradient centrifugation results of YOT027 Density gradient centrifugation results of Vph1-2flag ((a), (b), (c)), Nab2-2flag ((a),(b),(d)), Erg6-2flag ((a), (b), (e)), Emc1-4myc((f), (g), (h)), Pex3-4myc((f), (g), (i)), Cox4-12myc((f), (g), (j)), Chs5-4v5((k), (l), (m)), Anp1-4v5((k), (l), (n)), Snf7-4v5((k), (l), (o)), Pma1-3vsv((p), (q), (r)), 9vsv-Atg8((p), (q), (s)) of YOT027. GFP tagged proteins are set as control


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