The role of Yca1 in Proteostasis. Yca1 Regulates the Composition of the Insoluble Proteome.

ArticleinJournal of proteomics 81 · January 2013with16 Reads
Impact Factor: 3.89 · DOI: 10.1016/j.jprot.2013.01.014 · Source: PubMed
Abstract

Proteostasis, the process of balancing protein production with protein degradation is vital to normal cell function. Defects within the mechanisms that control proteostasis lead to increased content of a specialized insoluble protein fraction that forms dense aggregates within the cell. We have previously implicated the S. cerevisiae metacaspase Yca1 as an active participant in maintaining proteostasis, whereby Yca1 acts to limit aggregate content. Here, we further characterized the proteostasis role of Yca1 by conducting proteomic analysis of the insoluble protein fraction in wildtype and yca1 knockout cells, under normal and heat stressed conditions. Our findings suggest that the composition of insoluble protein fraction is non-specific and comprises a wide array of protein species rather than a limited repertoire of aggregate susceptible proteins or peptides. Interestingly, the loss of Yca1 led to a significant decrease of proteins that control ribosome biogenesis and protein synthesis within the insoluble fraction, indicating that the cell may invoke a compensatory mechanism to limit protein production during stress, a feature dependent on Yca1 activity. Finally, we noted that protein degradation factors such as Cdc48 co-localize with Yca1 to the insoluble fraction, supporting the hypothesis that Yca1 may act primarily to dissolve or reduce accumulated aggregates.

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Available from: Amit Shrestha, Jan 12, 2016
The role of Yca1 in proteostasis. Yca1 regulates the
composition of the insoluble proteome
Amit Shrestha
a, b
, Lawrence G. Puente
a
, Steve Brunette
a
, Lynn A. Megeney
a, b,
a
Ottawa Hospital Research Institute, Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital, Ottawa, Ontario,
Canada, K1H8L6
b
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada
ARTICLE INFO ABSTRACT
Available online 30 January 2013 Proteostasis, the process of balancing protein production with protein degradation is vital to
normal cell function. Defects within the mechanisms that control proteostasis lead to increased
content of a specialized insoluble protein fraction that forms dense aggregates within the cell.
We have previously implicated the Saccharomyces cerevisiae metacaspase Yca1 as an active
participant in maintaining proteostasis, whereby Yca1 acts to limit aggregate content. Here, we
furthercharacterizedtheproteostasisroleofYca1byconductingproteomicanalysisofthe
insoluble protein fraction in wildtype and Yca1 knockout cells, under normal and heat stressed
conditions. Our findings suggest that the compositionofinsolubleproteinfractionisnon-specific
and comprises a wide array of protein species rather than a limited repertoire of aggregate
susceptible proteins or peptides. Interestingly, the loss of Yca1 led to a significant decrease of
proteins that control ribosome biogenesis and protein synthesis within the insoluble fraction,
indicating that the cell may invoke a compensatory mechani sm to limit protein production
during stress, a feature dependent on Yca1 activity. Finally, we noted that protein degradation
factors such as Cdc48 co-localize with Yca1 to the insoluble fraction, supporting the hypothesis
that Yca1 may act primarily to dissolve or reduce accumulated aggregates.
This article is part of a Special Issue entitled: From protein structures to clinical applications.
© 2013 Elsevier B.V. All rights reserved.
Keywords:
Yca1
Cdc48
Proteostasis
Aggregates
Heat stress
1. Introduction
The regulation of protein content and solubility is an
indispensible feature which ensures the fidelity of key funda-
mental processes within the cell. Within this context the
endoplasmic reticulum (ER) provides the first oversight step to
ensure appropriate protein behavior, a unique environment
which is optimized for post-translational modifications and
proper folding of newly synthesized peptides. Disturbances
within the cell such as flaws during protein biogenesis,
environmental stresses and age related decline disrupt the
fidelity provided by the quality control mechanisms, allowing
for misfolded and/or damaged proteins to aggregate [1].
Accordingly, this accumulation of protein aggregates has been
associated with the progression and/or initiation of various
neurodegenerative diseases such as Alzheimer's, Huntington's,
Parkinson's, amyotrophic lateral sclerosis (ALS) as well as
various inclusion body myopathies [2,3].
Currently, it is well agreed upon that the extent of β-sheet
organization determines the structure of aggregates, which can
either be amorphous or amyloid in nature [4,5 ]. Indeed, exami-
nations of the morphology of protein aggregates suggest that
these structures are in fact ordered and arise from specific
interactions between misfolded protein intermediates [6].Specif-
ically, stretches of hydrophobic residues that are exposed in
conformers, may act as the seed to promote peptide/protein
JOURNAL OF PROTEOMICS 81 (2013) 24 30
This article is part of a Special Issue entitled: From protein structures to clinical applications.
Corresponding author at: Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
E-mail address: lmegeney@ohri.ca (L.A. Megeney).
1874-3919/$ see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jprot.2013.01.014
Available online at www.sciencedirect.com
www.elsevier.com/locate/jprot
Page 1
aggregation [7,8]. However, aggregation prone protein species can
also prompt numerous other protein species to aggregate or
confine them within the aggregate structures, suggesting that
aggregation is a proteome wide phenomenon [911]. In addition
to aggregate composition, there is considerable debate regarding
the cellular role of aggregates. The standard hypothesis suggests
that aggregate buildup is detrimental and a significant contrib-
utor to cellular dysfunction. In support of this supposition,
disease causing proteins (in a wide variety of neurodegenerative
diseases) are generally identified within aggregate material
[1214]. However, the alternative model suggests that the cell
sequesters non-functional proteins as a defense mechanism,
limiting exposure of the cell to an otherwise toxic structure [15].
The study of protein aggregation control has benefited from
the use of tractable model systems. In Saccharomyces cerevisiae,
aggregates are known to be sequestered and deposited in various
regions and can either undergo re-solubilization by the Hsp40/
Hsp70 bi-chaperone system and the AAA+ activity of Hsp104 or
are targeted for degradation by one of two ways; proteosomal
degradation or via autophagy [1618]. Furthermore, during
conditions of stress such as heat shock treatment, the
bi-chaperone system has been shown to be more im portant for
thermotolerance and re-solubilization rather than targeting
the aggregated proteins for degradation [1,19]. Recent evidence
suggests that proteases, such as the metacaspase Yca1, also play
a key role in non-death processes such as regulation of cell cycle
progression and maintaining proteostasis [20,21]. Specificall y,
Yca1 has been shown to condense into distinct discernible foci
which coincided with Hsp104 foci formation. Furthermore, Yca1
strongly associated with components of the bi-chaperone sys-
tem, namely Ssa1/2 (Hsp70) and Ydj1, as well as the small heat
shock protein Hsp42 and the AAA+ ATPase, Cdc48 [21]. Strikingly,
the loss of Yca1 expression or loss of Yca1 catalytic activity
resulted in an increase in insoluble protein content. Taken
together these observations suggest that Yca1 protease activity is
an indispensible component of the aggregate control machinery.
Here, we utilized the study of Yca1 function to further
delineate the mechanisms that control aggregate formation
and dissolution. In wildtype yeast, we noted that the insoluble
proteome was comprised of a large number of protein species
rather than a limited protein cohort. The loss of Yca1 led to a
significant decrease in ribosomal proteins and translational
control factors within the insoluble fraction, suggesting that
control over general protein production in response to aggre-
gate accumulation is disrupted with the removal of Yca1. In
addition, we noted that recruitment of the AAA+ ATPase Cdc48
to the insoluble protein fraction was strongly dependent on
Yca1 expression, whereas other recruitment of other compo-
nents of the chaperone system remained unaffected within the
insoluble protein fraction. We interpret this observation to
suggest that Yca1 protease activity is targeted to dismantle
protein aggregates rather than limit aggregate formation per se.
2. Materials and methods
2.1. Yeast strains and growth conditions
The wildtype BY4741 and Δyca1 strains of S. cerevisiae (Open
Biosystems) were grown in acidified YPD media (1% yeast extract,
2% peptone and 2% glucose, pH 3.5). 5 ml starter cultures of YPD
were inoculated with a single colony and grown overnight. Larger
YPD cultures were then inoculated from the starter cultures and
grown to mid-logarithmic phase (OD
600
0.50.6) at 30 °C with
orbital rotation. Cells were then collected via centrifugation at
2800 rpm for 5 min, washed with water and re-collected then
stored at 80 °C for later processing. For heat shock treatment,
mid-logarithmic cultures were further incubated at 42 °C for 1 h
before collection.
2.2. Protein extraction
Frozen cell pellets were suspended in modified RIPA buffer
(50 mM TrisHCl, 1 mM EDTA, 1% glycerol, 1% NP-40, pH 7.4)
containing protease inhibitors (Calciobiochem, Darmstadt,
Germany) and added to tubes containing 0.7 g of glass beads
(Sigma-Aldrich, Ontario, Canada). Cells were lysed using the
Disruptor Genie (Scientific Industries, New York, USA) at 4 °C
with alternating 1 min cycles of breaking and 1 min on ice for
a total of 12 min. The cell lysate was further cleared by
centrifugation at 2000 rpm for 1 min followed by 3000 rpm for
another minute. The final protein lysate was aliquoted and
stored at 80 °C.
2.3. Sample preparation
The total protein extracts were fractioned into soluble and
insoluble via centrifugation as described in [22].Equalamounts
of total protein extract were subjected to centrifugation at
15,000 g for 15 min. For the 2D LCMS analysis the supernatant
(soluble fraction) was discarded and the resulting protein pellet
(insoluble fraction) was further washed in modified RIPA buffer
and re-collected via centrifugation. The modified RIPA buffer
was discarded and the final pellet containing the insoluble
protein fraction was solubilized in buffer consisting of 8 M urea,
2% dithiothreitol and 50 mM TrisHCl pH 8.
2.4. SDS-PAGE and silver staining
The total protein extract was fractioned into soluble and
insoluble as described above. The insoluble protein pellet was
dissolved in modified RIPA buffer via vortex and loaded onto a
10% acrylamide gel containing SDS (0.1% w/v) with equal
volume of sample buffer. The electrophoretic separation of the
insoluble protein fraction was conducted at 100 V for 1014 h.
The corresponding soluble fraction was also separated similarly
on separate gels. After separation, the proteins were either
stained using the silver stain method described in [23] or
transferred onto a membrane for western hybridization [21].
2.5. Western hybridization
Proteins fractioned via SDS-PAGE were transferred to 0.45 μM
PVDF membrane (Millipore) on a TRANS-BLOT SD apparatus
(Bio-Rad). Membranes were blocked with TBST containing 5%
skim milk for minimum of 1 h after which they were
supplemented with primary antibody and further incubated
at 4 º C overnight. Bands were detected using primary
antibodies specific for Cdc48 (Thomas Sommer, Max Delbruck
Institute, Germany) as well as for Ydj1 and Ssa2 (Abcam) and
25JOURNAL OF PROTEOMICS 81 (2013) 24 30
Page 2
β-tubulin. Densitometry analysis of the resulting bands was
conducted using ImageJ software.
2.6. Protein digest
Proteins were reduced by addition of dithiothreitol and
alkylated by the addition of iodoacetamide before dilution of
the sample in 100 mM ammonium bicarbonate to reduce the
concentration of urea to<2 M. Proteins were digested using
trypsin (Promega). The resulting peptides were purified by
ZipTip (Milllipore), concentrated by Vacufuge (Eppendorf), and
resuspended in 0.1% trifluoroacetic acid.
2.7. 2D-LCMS/MS
Mas s spectral analysis was performed at the OHRI Proteomics
Core Facility (Ottawa, Canada). Peptides were analyzed by
2D-LCMS/MS on an LTQ Orbitrap XL hybrid mass spectrom-
eter with nanospray source (Thermo Scientific, USA) and an
UltiMate 3000 RSLC nano HPLC (Dionex). Peptides were loaded
onto a POROS 10S (Dionex) and eluted using ammonium
acetate salt steps (0 mM, 10 mM, 20 mM, 50 mM, 100 mM,
500 mM) onto a PepMap C18 trap column (Dionex) for 5 min
at 15 μl per minute, then eluted over a 60 min gradi ent of 3%
45% acetonitrile with 0.1% formic acid at 0.3 μlperminute
onto a 10-cm analytical column (New Objective Picofrit
self-packed with Agilent Zorbax C18), and nanosprayed into
the mass spectrometer. MS scans were acquired in the
Orbitrap module and MS
2
scans were acquired in the ion
trap module using data-dependent acquisition of the top 5
ions from each MS scan. Total data acquisition time=9 h.
Between samples, the system was washed three times with
1 M ammonium acetate salt injection on the SCX column and
a 60 min acetonitrile gradient over the C18 columns.
2.8. Protein identification using MASCOT
MASCOT 2.3.01 software (Matrix Science) was used to infer
peptide and protein identities from the mass spectra. The
observed MS/MS spectra were matched against S. cerevisiae
(6973 sequences) from the SwissProt database (version 57.15)
and also against 248 sequences from a Contaminants database
(downloaded from maxquant.org, June 9th 2011). Mass toler-
ance parameters were MS tolerance of ±5 ppm and MS/MS
tolerance of 0.6 Da. Enzyme specificity was set to Trypsin/P.
Oxidation of methionine, carbamidomethylation of cysteine,
protein N-terminal acetylation, deamidation, and/or conver-
sion of Glu or Gln to Pyro-Glu were allowed as variable
modifications. The emPAI scores reported by Mascot were
used as estimates of protein abundance. Mascot's Decoy Search
function was used to calculate False Discovery Rate (FDR).
2.9. Bioinformatics
Data was summarized and basic comparisons performed
using the Excel spreadsheet program (Microsoft). GO terms
annotation was performed using the Functional Annotation
Chart tool of the web service DAVID (Nature Protocols 2009;
4(1):44 & Genome Biology 2003; 4(5):P3). Relative enrichment of
GO terms was determined using the web service FunSpec
(http://funspec.med.utoronto.ca/).
3. Results
3.1. Composition of the insoluble protein fraction
Here, we used a proteomic approach to examine the
composition of the insolubl e p rotein fraction as a surrogat e
to model/understand protein aggregate formation in yeast.
The metacasp ase Yca1 has been implicated in regulating
levels of protein aggregates and hence we further validated
this hypothesis by comparing the composition of the insol-
uble protein fraction of the wildtype BY4741 (WT) to the Δyca1
(KO) strain under normal and heat stressed conditions by using a
two dimensional liquid chromatographytandem mass spec-
trometry (2D LCMS) approach which also provides the relative
protein abundance. In this study we altered our approach for
obtaining the insoluble fraction than reported previously in [20].
Initial experimentation of the insoluble fraction after conducting
the NP-40 detergent washes resulted in low yie ld with regards to
protein identification in comparison to what was observed from
a silver stained acrylamide gel of the same fraction (Fig. 1). We
postulated that the presence of NP-40 as well as glycerol in the
Fig. 1 Insoluble protein composition differs with induction
of heat stress. Silver stained gel depicting the protein profile
within the wildtype and knockout strains during normal
growth (A) and with heat stress treatment (B). Arrows
indicate the differences in the electrophoretic profile during
heat stress in comparison to normal growth.
26 JOURNAL OF PROTEOMICS 81 (2013) 24 30
Page 3
suspension buffer may be interfering with the 2D LCMS and
hence opted to use an alternate buffer that was known to be
compatible with this form of online MS analysis (see Materials
and methods). Importantly, similar results using this NP-40
buffer for insoluble protein isolation have been reported by other
groups for LCMS analysis [24]. Therefore, we reverted to the
original method as described in [22] where the insoluble fraction
was simply obtained by high speed centrifugation. Here, we
included an additional wash step with the same buffer used for
lysis and suspension and eliminated the NP-40 rich detergent
washes to minimize interference during the LCMS analysis.
Furthermore, this method yielded the insoluble fraction in its
entirety without any exclusion that may have resulted from
repeated detergent washes and have not been accounted for
previously.
Within the total 2120 proteins that were observed by the
MASCOT search engine, a proportion of these proteins were
exclusive to either condition. In the first data set we detected
678 proteins in normal growth conditions and 1178 proteins in
heat stress conditions for the wildtpe BY4741 strain. For the
Yca1 knockout strain we detected 880 proteins in normal
growth conditions and 1180 proteins during heat stress. In the
second data set we detected 573 proteins in normal growth
conditions and 876 proteins during heat stress for the BY4741
strain and 1181 proteins in normal conditions and 1015 proteins
during heat stress for the knockout mutant (Table S1). This
increase in proteins levels is similar to what we have previously
observed [21]. Furthermore, functional clustering using the
DAVID software resulted in the identification of 592 categories
for proteins within the insoluble fraction for the wildtype while
the Yca1 knockout showed 518 categories (Table S2). Addition-
ally, we also included the insoluble protein fraction of the
catalytically inactive mutant of Yca1, C297A, in our 2D LCMS
analysis and the corresponding data is also included in Table S1.
However, we chose to primarily focus on the data generated
from the wildtype and knockout strains for this study.
3.2. Enrichment analysis of proteins interacting with Yca1
Prior observations by our group suggest that Yca1 can interact
with Cdc48 and proteins of the Hsp40 and Hsp70 family [21].
Furthermore, these Yca1-interacting proteins are also active
members of protein re-solubilization/degradation machinery,
acting to limit the occurrence of misfolded proteins [1].
However, what remains unknown is whether these proteins
control aggregate deposition by remaining within the soluble
or insoluble fraction. For example, one may predict a number
of scenarios whereby the aggregate control machinery resides
within the soluble fraction of the proteome to limit aggregate
deposition. Alternatively, such Yca1 interacting proteins may
reside largely within the insoluble fraction to dissolve
aggregate composition. To address these alternate functional
scenarios, we examined the relative abundance of these
proteins during normal conditions and assessed enrichment
following the induction of heat stress. As shown in Fig. 2A, the
induction of heat stress led to a dramatic 9 fold increase in
levels of Cdc48 in the wildtype while the loss of Yca1 only
showed a modest 3 fold increase. Surprisingly, we did not
observe any significant increase in the levels of Ydj1, Ssa1 and
Ssa2, which are active components of the re-solubilization
machinery as well as for Ded1. We further verified the LCMS
data by western hybridization (Fig. 2B) and assessed the
change in levels of Cdc48, Ydj1 and Ssa2 following heat stress
by densitometry (Fig. 2C). We observed that Cdc48 levels were
reduced in the insoluble fraction of the knockout strain
compared to the wildtype while Ydj1 and Ssa2 levels were
similar in both strains, which are in agreement with the data
generated from LCMS. We also examined the changes in the
level of these proteins upon heat stress within the soluble
fraction. In both the wildtype and Yca1 knockout strains, Ydj1
and Ssa2 levels were observed to be similar between the
soluble and insoluble fractions. Interestingly, in the Yca1
knockout strain, the reduced level of Cdc48 observed in the
insoluble fraction was accompanied by an increased localiza-
tion of Cdc48 within the soluble fraction in comparison to the
wildtype. Thus, Cdc48 enrichment within the insoluble
fraction is dependent on the presence of Yca1.
3.3. Loss of Yca1 influences ribosomal protein function in
the insoluble fraction during stress
We have previously observed that the electrophoretic
profile of the insoluble protein fraction is variable and not
specific to a single or a few protein species [21]. To further
validate this observation we used the DAVID software to
retrieve the Gene Ontology (GO) terms associated with the
proteins that were observed to be enriched as a result of heat
shock in both Yca1 backgrounds. We noted that the GO term
ranking for processes relating to protein synthesis/translational
machinery showed a large decrease in the knockout strain
(Table 1). Thus, we furthered our analysis on four of the affected
GO terms; GO:0030686~90S preribosome, GO:0043039~tRNA
aminoacylation, GO:0043038~amino acid activation and
SP-PIR Ribosome biogenesis. We generated a non-
overlapping list of 73 proteins associated with these GO
terms that are affected by the induction of heat stress (Table S3).
To identify proteins that showed a fold increase in the wildtype,
suggesting a requirement for Yca1, we chose to highlight three
proteins of significant interest ERB1, NOP12 and YHR020W
(Fig. 3). ERB1 is a constituent of the 66S pre-ribosomal complex
required for the maturation of the 5.8S and 25S rRNAs [25].
NOP12 is a nucleolar protein involved in the large subunit
biogenesis and 25 s rRNA maturation [26]. YHR020W is an
uncharacterized essential protein in yeast which shares simi-
larity with proline-tRNA ligase and is postulated to interact with
ribosomes [27,28]. All three proteins were considerably enriched
during heat stress in the wildtype amounting to larger than
threefold, whereas within the knockout strains their levels
either remained the same or were reduced.
3.4. Prion protein levels are unaffected in Δyca1 cells
Interestingly in yeast, aggregates of prionogenic proteins,
such as [PSI
+
], are known to be cytoplasmically inherited by
daughter cells which ensure the transfer of epigenetic traits
[29]. To assess for a role of Yca1 in regulating levels of such
misfolded proteins we searched our protein list for known
prions that may be present in the insoluble protein fraction.
Our data generated from the 2D LCMS analysis included the
[NU+] prion protein form of NEW1 and the prion form of RNQ1
27JOURNAL OF PROTEOMICS 81 (2013) 24 30
Page 4
protein [PIN+]. We analyzed the relative abundance using
emPAI scores of [NU+] which is depicted in Fig. 4. Under
normal growth circumstances, [NU+] levels were more
abundant in the knockout strain. Consequently, the induction
of heat stress led to a reduction in [NU+] levels.
4. Discussion
In this study we identified the constituents of the insoluble
protein fraction in wildtype and Yca1 null backgrounds during
normal growth and heat stress. Surprisingly, the 2D LCMS
analysis led to the identification of over 2000 proteins within the
insoluble fraction. Although these proteins are not exclusive
components protein aggregates, this dataset suggests that the
physical prelude to aggregate formation i.e. deposition to an
insoluble protein compartment, is far more complex than has
been previously suggested. A cohort of these proteins within our
dataset could result from being confined within larger struc-
tures in the cell that resist solubilization but do not represent
true insoluble protein species, a feature that has been reported
previously [11]. Nonetheless, our approach implicates a wide
array of proteins that may be targeted for aggregation, under
normal conditions and stress. As such the dataset generated
from this study will serve as a useful tool for subsequent
investigations in this field.
To further validate the role of Yca1 in regulating cellular
aggregate levels, we assessed the expression of Yca1-interacting
that had been previously and indepen dently confirmed as
components of protein aggregate remodeling platforms. The
reduced levels of Cdc48 in the insoluble fraction and the
concomitant retention within the soluble fraction of the Yca1
null strain during stressed conditions suggests a dependency on
Yca1 to relocate Cdc48 to the insoluble protein fraction. Cdc48 is
an AAA+ ATPase and has been well characterized in its role in
ERAD factories as well as in the formation and clearing protein
aggregates [30,31]. Therefore, it may be reasonable to conjecture
that the presence of Cdc48 in the insoluble fraction may be to
perform a similar role, i.e. re-solubilization or targeting aggre-
gates for degradation. Interestingly, the other chaperone pro-
teins known to interact with Yca1 did not display a similar
Fig. 2 Cdc48 recruitment to the insoluble protein fraction is Yca1-dependent. (A) A graphical representation of the average fold
increase observed for each of the Yca1-interacting protein upon the induction of heat stress in the two strains obtained from
the 2D LCMS analysis (n=2; ±SEM). (B) Western blot showing the levels of Cdc48, Ydj1 and Ssa2 within the soluble and
insoluble fractions during normal (I) and heat stress (II) and the respective enrichment determined by densitometry analysis,
normalized to the wildtype (C, n = 3; ±SEM; SOL soluble, INS insoluble). β-tubulin levels in the respective soluble fraction
served as a loading control.
Table 1 GO term ranking for ribosomal and protein
synthesis related processes in wildtype and Yca1
knockout strains under heat stress.
Category Term Rank in
WT
Rank in
KO
GOTERM_CC_FAT GO-0030686_90S
preribosome
140 498
GOTERM_BP_FAT GO-0043039_tRNA
aminoacylation
91 325
GOTERM_BP_FAT GO-0043038_amino acid
activation
92 326
SP_PIR_KEYWORDS Ribosome biogenesis 62 264
28 JOURNAL OF PROTEOMICS 81 (2013) 24 30
Page 5
co-localization to the insoluble fraction. This observation may
indicate that the Yca1 and Hsp40/70 interactions are transient
and do not persist within the insoluble compartment.
Additionally, the reduction in [NU+] levels suggest that
during stress condit ions in Y ca1 null cells as well as wildtype
cells may reflect a disparity between prion biology and
proteostasis. Furthermore, of the numerous prions known in
yeast [32],wewereonlyabletodetecttwoinouranalyses,
which does not present us with sufficient data to further
investigate our speculation.
Our observation regarding alterations in rib osomal function
as a result of heat stress induction was unexpected. Our analyses
suggest that the ribosomal proteins listed in Table S2 are indeed
affected by the stressed condition leading to their localization
within the insoluble protein fraction and a proportion of these
proteins may depend on Yca1 for this to occur. Previous reports
in yeast suggest that cells favor re-solubilization over degrada-
tion of aggregates [1,19] Thus, it is tempting to speculate that
Yca1 function may also have impli cations on ribosome biogen-
esis and protein synthesis.
5. Conclusions
To further characterize the role of Yca1 in proteostasis we
conducted a 2D LCMS analysis of the insoluble protein
fraction in wildtype and Yca1 knockout cells. The resulting
analysis determined that the composition of the insoluble
protein fraction was non-specific and comprised a wide array
of proteins. Furthermore, Cdc48 levels within this fraction are
dependent on the presence of Yca1. Despite previous reports
our observations do not support the hypothesis that Yca1 has
a role in prion biology. However, our results suggest that loss
of Yca1 affects ribosomal protein function.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.jprot.2013.01.014.
Acknowledgments
We would like to thank Drs. Thomas Sommer and Ernst Jarosch
from the Max-Delbrueck-Center for Molecular Medicine for
providing the yeast anti-Cdc48 primary antibody. L.A.M. held
the Mach Gaensslen Chair in Cardiac Research. The work in the
laboratory of L.A.M. is supported by grants from the Canadian
Institutes of Health Research, the Muscular Dystrophy Associ-
ation and the Ontario Research Fund.
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30 JOURNAL OF PROTEOMICS 81 (2013) 24 30
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    • "On the other hand, the ability of metacaspase to both promote and antagonize different cell cycle checkpoints has been demonstrated, representing an early form of the proliferation/ differentiation regulatory activity exhibited by metazoan caspases [16]. YCA1 also contributes to the fitness and adaptability of growing yeast through clearance of insoluble protein aggregates [17] [18] and has been implicated in the regulation of antioxidant status and mitochondrial respiration [15] [19] [20]. The concept of non-apoptotic roles of metacaspase has been expanded in our previous work in which we have performed a comparative analysis between wild type and Δyca1 cells using combined proteomic and metabolomic approach. "
    [Show abstract] [Hide abstract] ABSTRACT: Caspase proteases are responsible for the regulated disassembly of the cell into apoptotic bodies during mammalian apoptosis. Structural homologues of the caspase family (called metacaspases) are involved in programmed cell death in single-cell eukaryotes, yet the molecular mechanisms that contribute to death are currently undefined. Recent evidence revealed that a programmed cell death process is induced by acetic acid (AA-PCD) in Saccharomyces cerevisiae both in the presence and absence of metacaspase encoding gene YCA1. Here, we report an unexpected role for the yeast metacaspase in protein quality and metabolite control. By using an "omics" approach, we focused our attention on proteins and metabolites differentially modulated en route to AA-PCD either in wild type or YCA1-lacking cells. Quantitative proteomic and metabolomic analyses of wild type and Δyca1 cells identified significant alterations in carbohydrate catabolism, lipid metabolism, proteolysis and stress-response, highlighting the main roles of metacaspase in AA-PCD. Finally, deletion of YCA1 led to AA-PCD pathway through the activation of ceramides, whereas in the presence of the gene yeast cells underwent an AA-PCD pathway characterized by the shift of the main glycolytic pathway to the pentose phosphate pathway and a proteolytic mechanism to cope with oxidative stress. The yeast metacaspase regulates both proteolytic activities through the ubiquitin-proteasome system and ceramide metabolism as revealed by proteome and metabolome profiling of YCA1-knock-out cells during acetic-acid induced programmed cell death. Copyright © 2015 Elsevier B.V. All rights reserved.
    Full-text · Article · Aug 2015 · Journal of Proteomics
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  • [Show abstract] [Hide abstract] ABSTRACT: The two metacaspases MCA1 and MCA2 of the fungal aging model organism Podospora anserina (PaMCA1 and PaMCA2, respectively) have previously been demonstrated to be involved in the control of programmed cell death (PCD) and life span. In order to identify specific pathways and components which are controlled by the activity of these enzymes, we set out to characterize them further. Heterologous overexpression in Escherichia coli of the two metacaspase genes resulted in the production of proteins which aggregate and form inclusion bodies from which the active protein has been recovered via refolding in appropriate buffers. The renaturated proteins are characterized by an arginine-specific activity and are active in caspase-like self-maturation leading to the generation of characteristic small protein fragments. Both activities are dependent on the presence of calcium. Incubation of the two metacaspases with recombinant poly(ADP-ribose) polymerase (PARP), a known substrate of mammalian caspases, led to the identification of PARP as a substrate of the two P. anserina proteases. Using double mutants in which P. anserina Parp (PaParp) is overexpressed and PaMca1 is either overexpressed or deleted, we provide evidence for in vivo degradation of PaPARP by PaMCA1. These results support the idea that the substrate profiles of caspases and metacaspases are at least partially overlapping. Moreover, they link PCD and DNA maintenance in the complex network of molecular pathways involved in aging and life span control.
    Full-text · Article · Apr 2013 · Eukaryotic Cell
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  • [Show abstract] [Hide abstract] ABSTRACT: How do cells age and die? For the past 20 years, the budding yeast, Saccharomyces cerevisiae, has been used as a model organism to uncover the genes that regulate lifespan and cell death. More recently, investigators have begun to interrogate the other yeasts, the fission yeast, Schizosaccharomyces pombe, and the human fungal pathogen, Candida albicans, to determine if similar longevity and cell death pathways exist in these organisms. After summarizing the longevity and cell death phenotypes in S. cerevisiae, this mini-review surveys the progress made in the study of both aging and programed cell death (PCD) in the yeast models, with a focus on the biology of S. pombe and C. albicans. Particular emphasis is placed on the similarities and differences between the two types of aging, replicative aging, and chronological aging, and between the three types of cell death, intrinsic apoptosis, autophagic cell death, and regulated necrosis, found in these yeasts. The development of the additional microbial models for aging and PCD in the other yeasts may help further elucidate the mechanisms of longevity and cell death regulation in eukaryotes.
    No preview · Article · Oct 2013 · FEMS Yeast Research
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