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Analyzing the Functionality of Non-native Hsp70 Proteins in Saccharomyces cerevisiae

酿酒酵母中非自身hsp70蛋白的功能分析

LK Laura E. Knighton
LS Lizbeth P. Saa
AR Adam M. Reitzel
AT Andrew W. Truman

发布: 2019年10月05日第9卷第19期 DOI: 10.21769/BioProtoc.3389 浏览次数: 3877

评审: Juan Facundo Rodriguez AyalaAnna A. ZorinaFernando A Gonzales-Zubiate

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Abstract

Yeast are an ideal system to study Heat Shock Protein 70 (Hsp70) function in a cellular context. This protocol was generated to analyze the function of non-native Hsp70 proteins by expressing them as the sole cytosolic Hsp70 in yeast. As an initial step, Hsp70 variants (such as Ssa1 point mutants and non-yeast versions such as Nematostella vectensis NvHsp70A, B and D) are cloned into an appropriate expression plasmid. Next, these plasmids are transformed into ssa1-4∆ yeast [expressing native Ssa1 from an uracil-based (URA3) plasmid] which are subsequently cured of the original yeast on 5-Fluroorotic Acid (5-FOA). The resulting cells can be screened for a variety of phenotypes which match to the activity of well-studied cellular pathways.

Keywords: Hsp70 (Hsp70) search Molecular chaperones (分子伴侣) search Nematostella (线虫病) search Plasmid swap (质粒互换) search

Background

cHsp70 is a molecular chaperone that plays a role in protein folding of newly synthesized and misfolded proteins (Rosenzweig et al., 2019). It also controls the activity of regulatory proteins that contribute to cell cycle progression, protein degradation, apoptosis and resistance to anticancer therapeutics. Hsp70 is highly conserved throughout nature and is essential for cell viability. Organisms can express several highly similar Hsp70 isoforms. For example, the budding yeast Saccharomyces cerevisiae expresses 4 cytosolic isoforms, Ssa1-4 (Lotz et al., 2019). In order to characterize Hsp70 function, we can express different Hsp70 isoforms, paralogs, point mutations and truncations as the sole cytosolic Hsp70 protein in S. cerevisiae using a 5-FOA plasmid swap strategy (Boeke et al.,1987).

We were recently able to use this methodology to express Nematostella vectensis Hsp70 isoforms (NvHsp70A, B and D) in yeast and asses their functionality through phenotypic screens, growth assays and analysis of interaction partners (Waller et al., 2018; Knighton et al., 2019). These results contribute to a better understanding of Hsp70 isoform function and the potential basis of local adaptation in populations of N. vectensis.

Materials and Reagents

  1. Pipette tips (10 μl, 300 μl, 1000 μl) (Neptune, catalog number: 2347; US Scientifica, catalog number: 1110-9700; Neptune, catalog number: 2167)
  2. Eppendorf 1.5 ml tubes (VWR, catalog number: 87003-294)
  3. 15 ml Falcon tubes (VWR, catalog number: 525-0449)
  4. 50 ml Falcon tubes (VWR, catalog number: 525-0447)
  5. Petri dishes (Thermo Fisher, catalog number: FB0875713)
  6. 96-well microplate (Greiner bio-one, catalog number: 655101)
  7. pAG415GPD-ccdB vector (Addgene plasmid # 14146; http://n2t.net/addgene:14146; RRID: Addgene_14146)
  8. Q5® High-Fidelity 2x Master Mix (NEB, catalog number: M0492S)
  9. ssa1-4∆ yeast strain (Jaiswal et al., 2011)
  10. Top 10 E. coli cells (Invitrogen, catalog number: C404003)
  11. Single-stranded carrier DNA (salmon sperm DNA, Solarbio, catalog number: D8030)
  12. Tryptone (US Biological Life Sciences, catalog number: C16050360)
  13. Ampicillin (Thermo Fisher, catalog number: 1159027)
  14. Yeast extract (US Biological Life Sciences, catalog number: C16091364)
  15. Peptone (HIMEDIA, catalog number: RM001)
  16. Glucose (VWR Amresco Life Science, catalog number: 0188)
  17. Agar (IBS Scientific, catalog number: IB49171)
  18. Adenine hemisulfate salt (Acros Organics, catalog number: 163631000)
  19. Yeast nitrogenous base (US Biological Life Sciences, catalog number: C16121501)
  20. Drop out mix minus leucine, methionine, uracil (US Biological Life Sciences, catalog number: C15050101)
  21. Drop out mix minus leucine (US Biological Life Sciences, catalog number: C15121094)
  22. Lithium Acetate (LiAc) (Sinopharm Chemical Reagent, catalog number: 30109760)
  23. Polyethylene glycol (PEG) (Sigma-Aldrich, catalog number: P3640)
  24. 0.05 g uracil (Arcos organics, catalog number: XLE-1000)
  25. 5-FOA (US biological life sciences, catalog number: 16052511)
  26. Hydroxyurea (HU) (Chem-implex Int’L INC, catalog number: 24533)
  27. Methyl methanesulfonate MMS (Acos organics, catalog number: 156891000)
  28. Sodium chloride (NaCl) (VWR, amrescolife science, catalog number: 0188)
  29. Cadmium chloride (CdCl2) (Acros organics, catalog number: 42350500)
  30. Hydrogen peroxide (H2O2) (Sigma-Aldrich, catalog number: 216763)
  31. Copper Chloride (CuCl2) (Sigma-Aldrich, catalog number: 222011)
  32. 2 ml of methionine (1 g/100 ml) (Arcos organics, catalog number: AC166160250) (see Recipes)
  33. YPDA Media (1 L) (see Recipes)
  34. Synthetic drop-out plates/media (1 L) (see Recipes)
  35. 5-FOA media (1 L) (see Recipes)
  36. Stock solution of 100 mM CuCl2 (see Recipes) 
  37. Stock solution of CdCl2 (see Recipes)
  38. Chemical plates (see Recipes)
  39. 1 M LiAc (500 ml) (see Recipes)
  40. 100 mM LiAc (500 ml) (see Recipes)
  41. 50% (w/v) PEG (500 ml) (see Recipes)

Equipment

  1. UV radiator (Spectroline UV Crosslinker, model: XLE-1000)
  2. 30 °C Incubator (VWR, catalog number: 89511-422)
  3. 37 °C incubator (VWR, catalog number: 89511-428)
  4. Heraeus Pico 21 Microcentrifuge (Thermo Fisher scientific, catalog number: 75002416)
  5. Heraeus Multifuge X1 Centrifuge Series (Thermo Fisher scientific, catalog number: 75004211)
  6. 30 °C Incubator Shaker (New Brunswick Excella E25)
  7. Thermomixer (Eppendorf, catalog number: 5382EJ909137)
  8. Heat block (Thermo Scientific, catalog number: 88870000)
  9. Thermal cycler (Bio-Rad, catalog number: 621BR26941)
  10. Replica plater for 96-well plate 8X6 Array (Sigma-Aldrich, catalog number: R2383-1EA) 
  11. Autoclave (Steris Amsco Cnetury, catalog number: SG-120)
  12. pH reader (RPI, catalog number: 850063)

Procedure

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文章信息

版权信息

© 2019 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Knighton, L. E., Saa, L. P., Reitzel, A. M. and Truman, A. W. (2019). Analyzing the Functionality of Non-native Hsp70 Proteins in Saccharomyces cerevisiae. Bio-protocol 9(19): e3389. DOI: 10.21769/BioProtoc.3389.

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分类

微生物学 > 异源表达系统 > 酿酒酵母

分子生物学 > 蛋白质 > 表达

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