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facilitate protein–protein interactions and contribute to the regulation
of proteolytic activity [ 6 , 7 ].
Metacaspase function in S. cerevisiae has been extensively
explored within a death-centric perspective, owing to the preexist-
ing view of caspase proteases as de facto mediators of programmed
cell death. Under such circumstances, various studies have shown
Yca1 involvement as an essential feature that progresses the apop-
totic phenotype induced by various insults [ 4 , 8 ]. However, obser-
vations of cell-death events independent of Yca1, such as the
inability of the proapoptotic hBax protein to engage Yca1 and
accelerate toxicity in yeast [ 9 ], argue against a death-only function
for Yca1. Importantly, the evolutionary retention of death-specifi c
machinery in single celled organisms, such as S. cerevisiae , supports
the premise that this protease may have evolved or coevolved non-
death functions [ 10 ].
Investigation of metacaspase biology in S. cerevisiae has since
elucidated “non-death” functions for Yca1, which may be shared
by other counterparts in trypanosoma and other yeast species [ 11 ].
Initially, a loss of function model demonstrated that Yca1 coordi-
nated processes that affect cell cycle timing and cellular fi tness;
Δyca1 cells displayed a delay in the G1/S transition and disruption
of microtubule assembly, via nocodazole exposure, did not halt
progression of Δyca1 cells through the G2/M checkpoint [ 7 ].
Subsequently, attempts to identify a molecular role for Yca1 under
these pro-survival parameters suggested that the protease func-
tions to ensure fi delity of protein turnover. Immunoprecipitation
[ 12 ] using the tandem affi nity purifi cation (TAP) method led to
identifi cation of Cdc48, Hsp40, and Hsp70 as high-affi nity inter-
action partners with Yca1 [ 13 ]. These Yca1-interacting proteins
are central contributors to cellular proteostasis; Cdc48 is involved
in ER-associated degradation and its level within the insoluble
proteome depends on the presence of Yca1 [ 14 ]; and Hsp40/70
together with Hsp100 constitute the “bi-chaperone” system that
actively re-solubilize misfolded proteins under stress conditions
[ 15 , 16 ]. Furthermore, fl uorescent microscopy studies showed
that Yca1 and Hsp104, a protein involved in aggregate dissolution
that serves as a marker for aggregated material, can co-localize in
cells collected from heat-stressed and aged cultures [ 13 ].
Additional comparisons between wild-type and Δyca1 cells
implicated Δyca1 cells with a protein turnover phenotype.
Sedimentation of cellular protein [ 17 – 19 ] to separate the insoluble
protein fraction followed by membrane fi ltration showed that
Δyca1 cells accumulate a greater amount of insoluble material [ 13 ].
In addition, analysis of vacuolar morphology via fl uorescence
microscopy and lipophilic styryl dyes [ 20 ] demonstrated that the
loss of Yca1 resulted in increased vacuole formation, possibly the
consequence of an increased protein aggregate load. Together,
these fi ndings support the hypothesis that Yca1 increases cellular
fi tness as a bona fi de player in cellular proteostasis.Amit Shrestha et al.http://www.ebook3000.com