Caspases,Paracaspases, and Metacaspases Methods and Protocols

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The discovery of a well-entrenched non-death function of

Yca1 has established metacaspases as versatile biological entities.

As such, the ability of Yca1 to cycle between death and non-death

activities demands stringent forms of analysis to accurately differ-

entiate the mechanisms that contribute to divergent cell fates. For

example, screening for known hallmark features of death such as

the integrity of the nucleus via DNA stains [ 13 ], may aid in delin-

eating processes that contribute to cell fate, yet these measures do

not distinguish the physiological roles of Yca1. This chapter con-

sists of the detailed protocols, which are instrumental in defi ning

and assessing the non-death, proteostasis function of the budding

yeast metacaspase Yca1.

2 Materials

1. YPD medium: 1 % (w/v) yeast extract, 2 % (w/v) Bacto
   Peptone, 2 % (v/v) dextrose, pH 3.5 ( see Note 1 ).


2. Orbital incubator (a range of 25–42 °C).


3. Buffer A: 50 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, 1 %
   (v/v) glycerol, 1 % (v/v) NP-40, pH 7.4. Store at 4 °C.


4. Buffer B: 50 mM Tris–HCl, 1 mM EDTA, 1 % (v/v) glycerol,
   0.1 % (v/v) NP-40, pH 7.4. Store at 4 °C.


5. Protease inhibitors (Calbiochem Cocktail Set IV or similar).


6. Acid washed glass beads, 0.4–0.6 mm in diameter (Sigma).


7. 27 G × ½ (0.4 mm × 13 mm) needles (Becton Dickinson).


8. 15 mL tubes (Sarstedt).


9. Buffer B: 50 mM Tris–HCl, 1 mM EDTA, 1 % (v/v) glycerol,
   0.1 % (v/v) NP-40, pH 7.4. Store at 4 °C.


10. Protease inhibitors (Calbiochem Cocktail Set IV or similar).


11. Wash buffer: 98 % (v/v) buffer B, 2 % (v/v) NP40.


12. Bio-Dot SF microfi ltration apparatus (Bio-Rad Labs).


13. Bio-Dot SF Filter paper (Bio-Rad Labs).


14. 0.45 μm PVDF membrane (e.g., Immobilon).


15. Buffer B: 50 mM Tris–HCl, 1 mM EDTA, 1 % (v/v) glycerol,
    0.1 % (v/v) NP-40, pH 7.4. Store at 4 °C.


16. Protease inhibitors (Calbiochem Cocktail Set IV or similar).


17. Wetting buffer: 80 % (v/v) buffer B, 20 % (v/v) methanol.


18. Coomassie Blue solution: 0.1 % (w/v) Coomassie Blue R-250,
    45 % (v/v) methanol, 10 % (v/v) acetic acid, 45 % (v/v) water.


19. Destain solution: 45 % (v/v) methanol, 10 % (v/v) acetic acid,
    45 % (v/v) water.

2.1 Yeast Growth
Conditions

2.2 Protein
Extraction

2.3 Sedimentation
Assay

2.4 Filter Trap Assay

Yca1 in Proteostasis