223Peter V. Bozhkov and Guy Salvesen (eds.), Caspases, Paracaspases, and Metacaspases: Methods and Protocols,
Methods in Molecular Biology, vol. 1133, DOI 10.1007/978-1-4939-0357-3_14, © Springer Science+Business Media New York 2014
Chapter 14
Monitoring the Proteostasis Function of the
Saccharomyces cerevisiae Metacaspase Yca1
Amit Shrestha , Robin E. C. Lee , and Lynn A. Megeney
Abstract
The functional versatility of metacaspase proteases has been established by reports of their involvement in
non-apoptotic cellular processes, in addition to their canonical role in apoptosis/programmed cell death.
While the budding yeast metacaspase Yca1 has been well characterized for its role in cell death regulation,
more recent examinations suggest that the protease may be involved in key processes that increase survival
and fi tness. More specifi cally, examinations suggest that Yca1 is central to maintaining cellular proteostasis
as it interacts with major components involved in protein biosynthesis and functions to limit aggregate
deposition. Here, we describe the methods utilized to analyze the role Yca1 in proteostasis.
Key words Aggregates , Filtration , Heat stress , Immunoprecipitation , Metacaspase , Proteostasis ,
Protocols , Vacuole morphology1 Introduction
Caspase enzymes, and related proteases, are broadly conserved
across the phyla of extant organisms. Structural investigations have
revealed that proteases within the clan CD family C14 share similar
quaternary architecture and an invariant cysteine–histidine cata-
lytic dyad [ 1 , 2 ]. In fungi, plants, and protozoa, such proteases are
termed metacaspases [ 3 ]. Metacaspases can be further subdivided
based on the presence or absence of a proline-rich N-terminal
prodomain, referred to as “type I” and “type II” classes respectively.
When compared across organisms, the number and type of meta-
caspases expressed is highly variable [ 4 ]. To date, only a single type
I metacaspase has been identifi ed in Saccharomyces cerevisiae [ 5 ].
This protease, termed Yca1 (also referred to as Mca1), harbors a
unique poly Q/N motif in its prodomain, upstream of its caspase-
like domain. The functional importance of this motif remains
largely unknown; however, it is suggested from metazoan initiator
caspases that the motifs within the prodomains of type I metacaspases