Caspases,Paracaspases, and Metacaspases Methods and Protocols

235

period with the stain. In order to avoid high background

staining, additional washes may be performed as described in

steps 11 and 12. For staining vacuoles through the stress-

recovery time course described in Subheading 3.1 , incubate

the cells with the stain for a minimum of 1 h before the 42 °C

for 1 h treatment.

13. Ethanol-fi xed cells can be stored for 36–48 h at 4 °C or for
    longer period (5 days) at −20 °C.

References

1. Aravind L, Koonin EV (2002) Classifi cation
   of the caspase–hemoglobinase fold: detection
   of new families and implications for the ori-
   gin of the eukaryotic separins. Proteins 46:
   355–367


2. Wong AH, Yan C, Shi Y (2012) Crystal struc-
   ture of the yeast metacaspase Yca1. J Biol Chem
   287:29251–29259


3. Uren AG, O’Rourke K, Aravind L et al (2000)
   Identifi cation of paracaspases and metacas-
   pases: two ancient families of caspase-like pro-
   teins one of which plays a key role in MALT
   lymphoma. Mol Cell 6:961–967


4. Tsiatsiani L, Van Breusegem F, Gallois P et al
   (2011) Metacaspases. Cell Death Differ 18:
   1279–1288


5. Madeo F, Herker E, Maldener C et al (2002) A
   caspase-related protease regulates apoptosis in
   yeast. Mol Cell 9:911–917


6. Carmona-Gutierrez D, Fröhlich KU, Kroemer
   G et al (2010) Metacaspases are caspases.
   Doubt no more. Cell Death Differ 17:377


7. Lee REC, Puente LG, Kaern M et al (2008) A
   non-death role of the yeast metacaspase:
   Yca1p alters cell cycle dynamics. PLoS ONE
   3:e2956


8. Madeo F, Carmona-Gutierrez D, Ring J et al
   (2009) Caspase-dependent and caspase-
   independent cell death pathways in yeast.
   Biochem Biophys Res Commun 382:227–231


9. Guscetti F, Nath N, Denko N (2005)
   Functional characterization of human pro-
   apoptotic molecules in yeast S. cerevisiae.
   FASEB J 19:464–466


10. Fernando P, Megeney LA (2007) Is caspase-
    dependent apoptosis only cell differentiation
    taken to the extreme? FASEB J 21:8–17
    11. Shrestha A, Megeney LA (2012) The non- death
    role of metacaspase proteases. Front Oncol 2:78
    12. Mitchell L, Lambert JP, Gerdes M et al (2008)
    Functional dissection of the NuA4 histone
    acetyltransferase reveals its role as a genetic hub
    and that Eaf1 is essential for complex integrity.
    Mol Cell Biol 28:2244–2256
    13. Lee REC, Brunette S, Puente LG et al (2010)
    Metacaspase Yca1 is required for clearance of
    insoluble protein aggregates. Proc Natl Acad
    Sci U S A 107:13348–13353
    14. Shrestha A, Brunette S, Puente LG et al (2013)
    The role of Yca1 in proteostasis. Yca1 regulates
    the composition of the insoluble proteome.
    J Proteomics 81:24–30
    15. Parsell DA, Kowal AS, Singer MA et al (1994)
    Protein disaggregation mediated by heat-shock
    protein Hsp104. Nature 372:475–478
    16. Glover JR, Lindquist S (1998) Hsp104,
    Hsp70, and Hsp40: a novel chaperone system
    that rescues previously aggregated proteins.
    Cell 94:73–82
    17. Cashikar AG, Duendald M, Lindquist SL
    (2005) A chaperone pathway in protein disag-
    gregation. Hsp26 alters the nature of protein
    aggregates to facilitate reactivation by Hsp104.
    J Biol Chem 280:23869–23875
    18. Haslbeck M, Miess A, Stromer T et al (2005)
    Disassembling protein aggregates in the yeast
    cytosol. The cooperation of Hsp26 with Ssa1
    and Hsp104. J Biol Chem 280:23861–23868
    19. Rand JD, Grant CM (2006) The thioredoxin
    system protects ribosomes against stress induced
    aggregation. Mol Biol Cell 17:387–401
    20. Journo D, Winter G, Abeliovich H (2008)
    Monitoring autophagy in yeast using FM4-64
    fl uorescence. Methods Enzymol 451:79–88

Yca1 in Proteostasis