New insights into cancer-related proteins provided by the yeast model

REVIEW ARTICLE

New insights into cancer-related proteins provided by theyeast modelClara Pereira, Isabel Coutinho, Joana Soares, Claudia Bessa, Mariana Leao and Lucılia Saraiva

REQUIMTE, Department of Biological Sciences, Laboratory of Microbiology, Faculty of Pharmacy, University of Porto, Portugal

Keywords

Bcl-2 family; cancer; caspases; humanized

yeast; p53; PKC family

Correspondence

L. Saraiva, Laboratory of Microbiology,

Faculty of Pharmacy, University of Porto,

Rua Anıbal Cunha 164, 4050-047 Porto,

Portugal

Fax: +351 222 003 977

Tel: +351 222 078 990

E-mail: [email protected]

(Received 3 October 2011, revised 28

December 2011, accepted 6 January 2012)

doi:10.1111/j.1742-4658.2012.08477.x

Cancer is a devastating disease with a profound impact on society. In

recent years, yeast has provided a valuable contribution with respect to

uncovering the molecular mechanisms underlying this disease, allowing the

identification of new targets and novel therapeutic opportunities. Indeed,

several attributes make yeast an ideal model system for the study of human

diseases. It combines a high level of conservation between its cellular pro-

cesses and those of mammalian cells, with advantages such as a short gen-

eration time, ease of genetic manipulation and a wealth of experimental

tools for genome- and proteome-wide analyses. Additionally, the heterolo-

gous expression of disease-causing proteins in yeast has been successfully

used to gain an understanding of the functions of these proteins and also

to provide clues about the mechanisms of disease progression. Yeast

research performed in recent years has demonstrated the tremendous

potential of this model system, especially with the validation of findings

obtained with yeast in more physiologically relevant models. The present

review covers the major aspects of the most recent developments in the

yeast research area with respect to cancer. It summarizes our current

knowledge on yeast as a cellular model for investigating the molecular

mechanisms of action of the major cancer-related proteins that, even with-

out yeast orthologues, still recapitulate in yeast some of the key aspects of

this cellular pathology. Moreover, the most recent contributions of yeast

genetics and high-throughput screening technologies that aim to identify

some of the potential causes underpinning this disorder, as well as discover

new therapeutic agents, are discussed.

Introduction

Most of our knowledge about basic cellular processes

has originated from model organisms [1]. The budding

yeast Saccharomyces cerevisiae has provided a major

contribution to fields as diverse as cell metabolism,

DNA replication, recombination, cell cycle, cell death,

protein folding, trafficking and organelle biogenesis

[2,3]. Unpredictably, in recent years, this knowledge

has been applied and expanded to an understanding of

human diseases. Indeed, as a cell model of human

diseases, yeast has provided insights into the basic pro-

cesses underlying pathogenesis. However, as a unicellu-

lar organism, the obvious limitation of this cell system

for the study of human diseases concerns an analysis

of the disease aspects that rely on multicellularity and

cell–cell interactions. Additionally, as a less complex

system, some relevant genes involved in the pathology

may not be present in the yeast genome. Although

important aspects of human diseases lie beyond the

Abbreviations

Apaf-1, apoptosis-inducing factor 1; cyt c, cytochrome c; FASAY, functional analysis of separated alleles in yeast; IAP, inhibitor of apoptosis

proteins; PFT, pifithrin; PKC, protein kinase C; ROS, reactive oxygen species; wt, wild-type.

FEBS Journal 279 (2012) 697–712 ª 2012 The Authors Journal compilation ª 2012 FEBS 697

reach of S. cerevisiae, this cell system has already

proved its value as a first-line tool in the discovery of

mechanistic processes involved in the disease. This has

been possible because molecular interaction networks

are unpredictably conserved from yeast to humans

[2,4]. A remarkable example is the direct application of

fundamental knowledge of cell cycle regulation, as

uncovered in yeast, towards studies in human cancer

biology [5]. Furthermore, with the finding that yeast

can undergo an apoptotic cell death exhibiting pheno-

typic features and basic molecular machinery similar

to those found in higher eukaryotes, the mechanisms

of apoptosis could also be intensively addressed in

yeast and the knowledge obtained transposed to

human cells, providing clues towards an understanding

of apoptosis-related diseases [6]. Yeast shows other

undeniable advantages as a model organism as far as

molecular studies are concerned. The yeast genome

was the first eukaryotic genome to be sequenced [7]

and, over the years, this knowledge has fueled whole-

genome scale screening methods, including microarrays

[8–10], two-hybrid analysis [11,12] and the use of dele-

tion and overexpression libraries [13,14]. The yeast

toolbox can also be used to unravel potential rescuing

mechanisms because it is particularly well-suited to

genetic suppressor isolation and chemical library

screening [3,15]. In addition, yeast presents many tech-

nical advantages over other systems, such as a short

generation time, ease of manipulation and a high ame-

nability to genetic modifications.

The advantages presented above have fuelled the

emergence of yeast models for several human dis-

eases, including cancer. When establishing cancer-

related protein models, different approaches are

adopted depending on the degree of conservation of

the protein under study. If the gene codifying for the

protein is conserved in yeast (e.g. TOR1) [16], it is

possible to directly study its function. If the gene

has no orthologue in yeast, the heterologous expres-

sion of the human gene in this organism (the so-

called ‘humanized yeast’) can still be highly informa-

tive because yeast may conserve protein interactions

that give clues to its function and pathobiology.

Yeast expressing the tumour suppressor p53 repre-

sents an example of this strategy [17]. The absence

of orthologues of a protein or an entire pathway can

sometimes even be advantageous because the protein

can be studied in a simpler eukaryotic environment,

without the interference of other proteins with simi-

lar or overlapping functions, as well as its endoge-

nous regulators. For example, yeast has been used

for the independent analysis of each isoform of the

protein kinase C (PKC) family [17,18]. Typical

approaches to study human proteins in yeast are pre-

sented in Fig. 1.

The present review focuses on the use of ‘humanized

yeast’ as a model system for studying the major human

proteins involved in cancer.

Caspase family members

It became evident that cellular and biochemical fea-

tures of apoptosis are a consequence of the cleavage of

a subset of proteins by proteases of the caspase family.

Caspases are a conserved family of cysteine-dependent

aspartate-specific proteases consisting of at least 15

members that can be divided into pro-apoptotic and

pro-inflammatory subfamilies. The pro-apoptotic casp-

ases can be further separated into activator or initiator

caspases (caspase-2, -8, -9, -10 and -12) and execu-

tioner or effector caspases (caspase-3, -6 and -7) that

are activated by the initiator caspases. It must be

noted, however, that this classification is an oversim-

plification because there are situations where pro-apop-

totic caspases (e.g. caspase-3) can mediate other

responses than apoptosis, including cell differentiation

and the activation of survival pathways [19]. Deregula-

tions in the expression or activity of these proteases

can lead to the development of several human apopto-

tic diseases, including cancer and neurodegenerative

disorders. Hence, an understanding of the cellular

function of caspase family members and the mecha-

nisms behind their specificity and regulation has been

the focus of extensive research [19,20].

The high complexity of mammalian caspase-signal-

ling pathways led several research groups to investigate

simpler eukaryotic systems as complementary cell

models. Although yeast encodes a metacaspase

(Yca1p) that shares structural homology and mecha-

nistic features with mammalian caspases, major differ-

ences in the primary cleavage specificity have lead to

the questioning of its classification as a ‘true’ caspase.

This has been the subject of an intensive discussion

[6,21]. Indeed, Yca1p has been implicated in the same

cellular processes as mammalian caspases, namely in

apoptosis [22]. However, although mammalian caspas-

es specifically cleave their substrates after aspartic acid

residues, metacaspases specifically cleave substrates

after an arginine or lysine (basic residues) [23].

Although it still remains a controversial issue,

recent studies have demonstrated that the activity of

mammalian caspases in yeast (namely of caspase-2, -3,

-4, -7, -8 and -10) is independent of Yca1p [24,25].

Yeast has therefore been widely exploited for the

independent analysis of several members of the

human caspase family with the aim of identifying their

Yeast research on cancer-related proteins C. Pereira et al.

698 FEBS Journal 279 (2012) 697–712 ª 2012 The Authors Journal compilation ª 2012 FEBS

regulators and substrates. For example, a modified

yeast two-hybrid system, based on the detection of

b-galactosidase activity, was used to identify caspase-3

[26] and -7 [27] substrates. An approach to monitor

the caspase activity was also developed in yeast using

a reporter system consisting of a transcription factor

linked by caspase cleavage sites to the intracellular

domain of a transmembrane protein. Caspase activa-

tion induced the release of the transcription factor

from the membrane, which in turn drove the transcrip-

tional activation of a reporter gene, such as bacterial

lacZ, therefore resulting in b-galactosidase activity

dependent on caspase activation [28,29]. However, the

most commonly used strategy for studying human

caspases in yeast has relied on the fact that high

expression levels of a caspase lead to a pronounced

yeast growth arrest. Unlike caspase-1, -2, -4, -5, -8, -10

and -13, caspase-3, -6, -7 and -9 do not auto-activate

when expressed in S. cerevisiae. Despite this, the

activation of these caspases in S. cerevisiae could be

achieved using different strategies as natural caspase

activators, such as other caspases or the apoptosis-

inducing factor (Apaf-1) (Fig. 2A). Indeed, the activa-

tion of caspase-3 in S. cerevisiae was successfully

achieved by co-expression with caspase-8 or -10, which

are two caspases that are efficiently processed and acti-

vated in yeast. Interestingly, these caspases were insuf-

ficient to catalyse the maturation of caspase-6. These

results distinguished sequential modes of action for dif-

ferent caspases in vivo [30]. Additionally, although

co-expression of caspase-9 and -3 in yeast had no cyto-

toxic effects, when the Apaf-1 was also expressed, a

pronounced yeast cell death was obtained. This

suggested the activation of caspase-9 by Apaf-1, which

in turn activates caspase-3. Accordingly, the possibility

of reconstituting the Apaf-1-activated pathway in yeast

was demonstrated [31]. Auto-activation of caspases

in yeast can also be obtained by using differently

Fig. 1. Typical approaches when studying human disease-related proteins in yeast. First, the gene of interest is cloned into a yeast vector with

a constitutive or more often regulatable promoter because many disease-related proteins are toxic when expressed in yeast. Second, the cyto-

toxicity of a human protein is determined by optical density and colony-forming unit (cfu) counts. The optical density is a simple and fast

method that can be used in genomic- and pharmacological-wide screens. The cfu counts allow distinction between decreased growth and

increased cell death. When the human protein is involved in ageing or stress pathways, it may be needed to reproduce these conditions with

yeast when assaying for protein function to uncover a phenotype. Third, whether the human protein-induced growth inhibition is the result of

cell cycle arrest, stress or cell death can be assessed. Several techniques are available to distinguish between different types of cell death

(apoptotic, necrotic or autophagic). Apoptosis can be identified for instance by assessing DNA fragmentation using terminal deoxynucleotidyl

transferase dUTP nick end labelling, chromatin condensation upon staining with 4¢,6-diamidino-2-phenylindole, and externalization of phosphati-

dilserine (PS) using Annexin V stainning. Propidium iodide, which only stains cells with ruptured cellular membranes, is used as a marker of

necrosis. Autophagy can be assessed by monitoring the increase in mature Atg8p or the formation of autophagosomes by electron micros-

copy. Because many proteins affect vesicular trafficking, this pathway can be monitored by monitoring the uptake of the dye FM4-64. As a

first-line model, all the discoveries made in yeast must be validated in more physiological models of the disease.

C. Pereira et al. Yeast research on cancer-related proteins


engineered auto-activated caspase variants (Fig. 2B–E).

The caspase activity in yeast has been frequently asso-

ciated with the induction of a cell death phenotype.

Indeed, several studies have explored this feature for

the functional analysis of human caspase-3, -8 and -10,

as well as for the identification of yeast specific protein

interaction partners and corresponding orthologues in

human cells [24,25]. However, it was found that the

phenotypic consequences of the expressed human cas-

pase in yeast were highly dependent on the yeast strain

used. This is probably a result of differences in the

expression levels of endogenous caspase substrates

and ⁄or of other proteases with similar functions

among the yeast strains [24,32]. Moreover, the strategy

aiming to produce auto-activating forms of caspases

was also referred to as having some impact on the final

outcome of caspase activation in yeast. Indeed, differ-

ent efficiencies on generating an active caspase can be

related to different proteolytic activities. Although the

co-expression of caspase-3 subunits as separate

proteins caused yeast growth inhibition without the

induction of cell death [33,34], the expression of the

reverse-caspase-3 caused necrotic yeast cell death [24].

However, under our experimental conditions, the

expression of a reverse form of human caspase-3 in yeast

lead to a pronounced growth arrest [35] associated with

an increase in DNA fragmentation and mitochondrial

reactive oxygen species (ROS) production, as well as the

maintenance of plasma membrane integrity (I. Coutinho

and L. Saraiva, unpublished data). These results there-

fore indicate the induction of apoptotic (instead of

necrotic) yeast cell death by human reverse caspase-3.

Many aspects of the regulation of mammalian cas-

pases by natural adapters could also be recapitulated

in yeast. For example, it was shown that the cytotoxic

effect of caspase-8 in S. cerevisiae [30] and of caspase-3

in Schizosaccharomyces pombe [32] was abrogated by

the co-expression of the pan-caspase inhibitor baculo-

virus protein p35. Additionally, it was reported that

inhibitors of apoptosis proteins (IAPs) in mammals,

namely XIAP, suppressed the growth defect of S. cere-

visiae expressing active human caspase-3 [34]. A subse-

quent study showed that the protective effect of IAPs

could be abrogated by the co-expression of Drosophila

pro-apoptotic proteins HID and GRIM or the mam-

malian protein DIABLO ⁄Smac [31]. Taken together,

these studies opened the possibility of using yeast-

based caspase assays to screen for chemical and genetic

Fig. 2. Different strategies of human caspase-3 activation in S. cerevisiae. (A) Natural caspase activators, namely caspase-8 ⁄ -10 and Apaf-1.

(B–E) Engineered auto-activated caspase variants that undergo spontaneous proteolytic processing or folding (also valid for caspase-6, -7

and -9). (B) Generation of reverse caspase, in which the small subunit precedes its prodomain and large subunit [27,30,31]. (C) Removal

of the N-terminal prodomain from the caspase coding sequence [31]. (D) Separately co-expressing the large and small subunits of an

active caspase [26]. (E) Joining in-frame the caspase cDNA to the coding regions for Escherichia coli b-galactosidase (lacZ ) [36]. Acti-

vated caspases lead to yeast growth arrest, which can be abolished by co-expression with p35 or IAPs, and by the small molecule cas-

pase-3 inhibitor Ac-DEVD-CMK. PD, prodomain; LS, large subunit; SS, small subunit.



inhibitors of caspase family members. Indeed, several

yeast assays have recently been developed for the high-

throughput screening of modulators of human caspases,

using either chemical or cDNA libraries [29]. Native cell

lysates from S. cerevisiae [24,30,32] and S. pombe [36]

expressing a human caspase were used to establish a

correlation between the effects of caspase expression on

yeast growth and the proteolytic processing and enzy-

matic activity of these caspases. Accordingly, a yeast

caspase-3 phenotypic assay, based on the measurement

of yeast cell growth, was developed to search for small

molecule inhibitors of this human caspase. In this assay,

the correlation established between the reversion of cas-

pase-3-induced yeast growth inhibition by a caspase-3

inhibitor (e.g. Ac-DEVD-CMK) and caspase-3 inhibi-

tion led to the discovery of new inhibitors of caspase-3

among a chemical library of vinyl sulfones [35]. The

exploitation of these assays to screen for activators and

inhibitors of individual caspase family members may

help in the discovery of new therapeutic agents against

cancer and neurodegeneration, respectively.

Bcl-2 family proteins

Bcl-2 family proteins include regulators of mitochon-

drial apoptotic signal transduction and, as such, have

an important role in diseases such as cancer. The

approximately 30 Bcl-2 family members can be classi-

fied, based on their different functions and correspond-

ing BH domains, as both anti-apoptotic (e.g. Bcl-2,

Bcl-xL and Mcl-1) and pro-apoptotic (e.g. Bax, Bak

and BH3-only) proteins [37].

The co-existence of several members of the Bcl-2

family and the complex interactions and regulatory

mechanisms in mammalian cells has hindered our

knowledge about this family of proteins and, subse-

quently, about the emergence of associated diseases. In

1994, Sato et al. [38] found that the chimeric protein

LexA-Bax was able to kill yeast, an effect that (as in

mammals) could be prevented by the anti-apoptotic

proteins Bcl-2 and Bcl-xL. Because Bcl-2 family pro-

teins were able to conserve at least part of their func-

tions when expressed in yeast, to overcome the

problem of mammalian redundant pathways, this

organism was selected by many groups for functional

analysis. It is important to note that, although yeast

was previously considered to be devoid of Bcl-2 family

members, a Bcl-xL interacting protein harbouring a

Bcl-2 homology (BH3) domain (Ybh3p) was recently

identified in S. cerevisiae [121].

The observation that Bax also inserted into yeast

mitochondrial membrane and caused cytochrome c

(cyt c) release [39], a hallmark of Bax function in

mammals, opened the way for the use of yeast in sev-

eral studies concerning this process [40]. For example,

several mitochondrial proteins, which were suspected

to be required for Bax activity in mammals, have

yeast orthologues and their roles were investigated in

this model. Although the requirement of some pro-

teins such as adenine nucleotide translocator is still

considered controversial [41,42], F0F1-ATPase appears

to be required for Bax toxicity in yeast [43,44]. An

additional controversial issue concerns the type of

Bax-induced yeast cell death (apoptotic versus auto-

phagic). Some features in yeast cells expressing Bax,

such as cyt c release from mitochondria [39], DNA

fragmentation and phosphatidylserine exposure [45],

supported an ‘apoptotic-like’ cell death involving the

formation of an outer membrane mitochondrial apop-

tosis-induced channel, which allows cyt c release and

is associated with oxidative stress and apoptosis

induction [45,46]. Yet, the discovery that cyt c release

was not essential for Bax-induced cell death, as well

as the presence of autophagic markers (namely Atg8p

maturation, vacular uptake of material and accumula-

tion of autophagosomes), suggested a Bax-induced

autophagic cell death [47,48]. Bax induction of auto-

phagy was recently reported in mammalian cells [49].

The function of pro-apoptotic (as Bax and Bid) or of

anti-apoptotic (as Bcl-xL and Bcl-2) proteins depends

on their ability to translocate, oligomerize and insert

into the mitochondrial membrane. Structural studies

in yeast gave rise to the first descriptions of BH

domains [50], as well as to the first insights into

domains and residues that are critical for mitochon-

drial insertion and toxicity [51,52]. For example,

although ablation of the Bax C-terminal domain was

found to be dispensable for mitochondrial localization

and cyt c release, the absence of the C-terminal

domain of Bcl-xL prevented this protein from rescu-

ing the cells from the effects of Bax [52]. Nevertheless,

heterodimerization was not crucial for Bcl-xL inhibi-

tion of Bax toxicity [53,54]. Such studies, along with a

previous study showing that Bcl-2 also prevented Bax-

induced cell death independent of heterodimerization,

provided the first evidence for heterodimerization-

independent mechanisms of Bcl-2 family proteins

[53,55].

The role of post-translational modifications in the

regulation of Bcl-2 family members has been a subject

of increased interest. Using yeast, it was observed that

some phosphorylatable serine residues of Bax were

able to affect its translocation to mitochondria, regu-

lating the release of cyt c and a triggering of the

apoptotic cascade [51]. Similarly, it was shown that

the cytoprotective effect of Bcl-xL in yeast cells



undergoing apoptosis was differently regulated by

PKC isoforms through modulation of the Bcl-xL phos-

phorylated state [18] (as discussed below).

The cytotoxicity of Bax in yeast allowed the screen-

ing for toxicity suppressors as putative anti-apoptotic

factors. The success of this approach was demon-

strated by the identification of new mammalian apop-

totic regulators, such as Bax inhibitor-1 [56],

bifunctional apoptosis regulator [57] and Calnexin or-

thologue Cnx1 [58].

Overall, studies on Bcl-2 family proteins in yeast,

mainly focussing on interactions among their members

and structure–activity relationships, have provided

important insights into the biology of these proteins.

Despite the amenability of yeast to high-throughput

screenings, only one study has used yeast for screening

a library of BH3 peptides specific for Mcl-1 or Bcl-xL

[59].

PKC family

The PKC family consists of at least 10 serine ⁄ threo-nine protein kinases grouped into three major PKC

subfamilies according to their primary structure and

the cofactors required for activation: classical (cPKCs;

a, bI, bII and c), novel (nPKCs; d, e, g and h) and

atypical (aPKCs; f and k\i). PKC isoforms are crucial

regulators of cell proliferation and death. Conse-

quently, it is not unexpected that the activity and

expression of some of these kinases are altered in can-

cer. PKC isoforms therefore represent promising thera-

peutic targets for the treatment of this pathology

[60,61].

A striking feature is that individual PKC isoforms

can exert either similar or opposite effects in cell pro-

liferation and death. PKCa, e and f have been mostly

described as promoters of cell proliferation and sur-

vival, and PKCd as a cell death promoter. However,

these roles have been widely questioned. Indeed,

depending on the biological context, overlapping func-

tions of a same PKC isoform in both promoting and

inhibiting cell proliferation and death have been fre-

quently reported [60,61]. The complexity of the PKC

family (i.e. the coexistence of several PKC isoforms in

a same cellular environment and the different expres-

sion profiles of PKC isoforms in different cell types)

has contributed to such contradicting reports. An indi-

vidual analysis of each PKC isoform would certainly

contribute to a clear understanding about their biology

and the identification of isoform-selective pharmaco-

logical modulators. This would help the development

of new therapeutic strategies based on the selective

modulation of these kinases. However, with respect to

the limitations presented above, the exclusive use of

mammalian cells would certainly hamper the achieve-

ment of such a goal.

In 1993, several studies performed by Riedel and

colleagues demonstrated that yeast, (endogenous PKC,

Pkc1p in S. cerevisiae, is a structural but nonfunctional

homologue of mammalian PKC isoforms [62,63]),

comprised a promising cell system for studying individ-

ual mammalian PKC isoforms [64–67]. These studies

showed that mammalian cPKCa and bI were function-

ally expressed in yeast, leading to biological responses

similar to those observed in mammalian cells. Indeed,

the phorbol ester activation of PKCa and bI caused

PKC down-regulation, uptake of extracellular Ca2+,

Ca2+-dependence of cell viability, changes in cell mor-

phology and an increase in the cell doubling time.

Taken together, these findings indicated the conserva-

tion in yeast of a mammalian PKC signalling pathway.

The observation that activation of a mammalian PKC

isoform in yeast induced a specific phenotype (i.e. an

increase in the cell doubling time that was propor-

tional to the level of its enzymatic activity) led to the

establishment of a yeast PKC phenotypic assay. As a

sensitive and fast method for quantitatively measuring

PKC activity, this yeast assay was successfully used for

functional, molecular and pharmacological studies of

mammalian PKC isoforms.

In a continuous effort to establish a PKC struc-

ture–function relationship, the yeast PKC assay has

been extensively used. For example, using this assay,

the role of cysteine-rich binding sites in the conserved

C1 region of the PKC regulatory domain, as well as

in the regulation of wild-type (wt) and mutants

PKCa by several activators (e.g. 4b-phorbol l2-myri-

state 13-acetate, mezerein and indolactam V), was

investigated [68,69]. Moreover, scanning mutagenesis

studies performed in yeast helped in the identification

of major regions within the PKCa regulatory domain

that are important for phorbol binding and phorbol-

dependent activation of the enzyme [70]. Additionally,

deletion analysis of PKCa in yeast allowed the identi-

fication of a novel regulatory segment in the C2

region, described by amino acids 260–280, which,

similar to the pseudosubstrate region, regulates PKCaactivity by preventing its constitutive activation [71].

The observation that PKCa activity was not strictly

regulated by the pseudosubstrate sequence led to

other studies investigating the regions within the regu-

latory domain essential for PKCa auto-inhibition [72],

which provided new data about the existence of intra-

molecular interactions between the regulatory and cata-

lytic regions of PKCa that maintain the enzyme in

its inactive conformation, thus regulating its catalytic



activity and the physical access of PKC modulators to

its target sites.

The yeast PKC phenotypic assay was also exten-

sively used in the characterization of the potency and

selectivity of known PKC activators [73] and inhibitors

[74,75], as well as in the screening for new potent and

selective PKC pharmacological modulators of individ-

ual mammalian PKC isoforms [74,76–78]. More

recently, using yeast cells expressing PKCa, bI, d, e or

f, a new potent and selective activator of nPKCd and

e was discovered [79]. The applicability of this small

molecule as an analytical probe for studying nPKCdand e cellular signalling pathways has been confirmed

in mammalian cells [80]. Furthermore, the stimulation

of an apoptotic pathway independent of anti-apoptotic

PKC isoforms (a, bI and f), as revealed by a study in

yeast [79], also supports the promising application of

coleon U as an anticancer drug. Interestingly, it was

also shown in yeast that, although 4b-phorbol l2-myri-

state 13-acetate activated nPKCd and e, inducing their

translocation from the cytosol to the plasma mem-

brane and G2 ⁄M cell cycle arrest, coleon U induced

the translocation of these isoforms to the nucleus and

metacaspase- and mitochondria-dependent apoptosis

[79]. These results corroborated those obtained in

mammalian cells showing that distinct stimuli can

induce the translocation of a specific PKC isoform to

distinct subcellular compartments, which was subse-

quently associated with distinct cellular responses [81].

Indeed, it was shown that the cellular localization of

PKCd regulates the survival ⁄death pathway. Although

the retention of PKCd in the cytoplasm is compatible

with cell survival, its nuclear retention is required for

commitment to apoptosis [82,83]. Consistently, the

nuclear targeting of kinases such as PKCd is consid-

ered as a new and essential regulatory mechanism that

directly influences the induction of apoptosis [82].

Thus, a broader outcome of this type of study was the

validation of this cell model to unravel intra-organelle

communication systems and their roles in the PKC iso-

form apoptotic signalling network.

The finding that yeast can undergo apoptosis with

characteristics similar to mammalian cells [6] uncov-

ered the possibility of exploiting this model organism

for unravelling the role of individual mammalian PKC

isoforms in the regulation of this cellular process, par-

ticularly of key apoptotic proteins such as Bcl-xL [18],

Bax [84] and p53 [17,85].

Using yeast cells co-expressing an individual mam-

malian PKC isoform and the anti-apoptotic Bcl-xL

protein, it was shown that PKC isoforms differently

regulated Bcl-xL anti-apoptotic activity in acetic acid-

induced yeast cell death by affecting its phosphorylated

state. Although PKCd had no effect on Bcl-xL activity,

PKCa abolished its anti-apoptotic effect through an

increase of the Bcl-xL phosphorylated form. On the

other hand, PKCe and f enhanced the Bcl-xL activity,

decreasing the Bcl-xL phosphorylated form. Consistent

with the results obtained in mammalian cells, the

results obtained in yeast showed that Bcl-xL phosphor-

ylation disables its anti-apoptotic function (Fig. 3). Du

et al. [86] proposed the existence of a kinase and phos-

phatase system in mammalian cells that may be oper-

ating in tandem, leading to a coordinated

phosphorylation–dephosphorylation cycle that modu-

lates Bcl-xL activity. However, the precise mechanisms

for this modulation remain undetermined. The study

in yeast provided new insights into the role of phos-

phorylation on the modulation of the Bcl-xL function,

identifying individual PKC isoforms as regulators of

the phosphorylation–dephosphorylation state of

Bcl-xL (Fig. 3). Additionally, it corroborated several

studies with respect to the identification of Bcl-2 family

members as major apoptotic targets of PKC isoforms

[87]. Indeed, it was recently demonstrated that PKCaalso regulates Bax in yeast [84]. The pro-apoptotic

activity of this Bcl-2 family protein depends on its

ability to translocate, oligomerize and insert into the

mitochondrial membrane after stress [40]. Using yeast

cells co-expressing Bax and PKCa, it was shown that

PKCa increased the translocation and insertion of

Bax into the outer mitochondrial membrane. This

was associated with an increase in cyt c release, ROS

production, mitochondrial network fragmentation and

Fig. 3. Regulation of Bcl-xL activity and phosphorylated state by

mammalian PKCa, e and f. PKCa abolished the Bcl-xL anti-apoptotic

activity by Bcl-xL phosphorylation, directly or through activation of

other kinase. PKCe and f enhanced the Bcl-xL anti-apoptotic activity

most likely through phosphorylation and, consequently, the activa-

tion of a phosphatase responsible for the decrease of the Bcl-xL

phosphorylated form.



autophagic cell death. Curiously, this PKCa effect was

revealed to be independent of its kinase activity. This

suggests that PKCa can promote apoptosis by a

kinase-independent way [84].

By contrast to human wt p53, which induces yeast

growth arrest when expressed in yeast, in the absence

of an exogenous activator (e.g. phorbol ester), PKCa,d, e and f do not interfere with yeast cell growth [22].

Using yeast cells co-expressing PKCa, d, e or f and wt

p53, the role of individual PKC isoforms in the regula-

tion of p53 activity was studied. The results obtained

revealed a differential regulation of p53 activity and

phosphorylation state by PKC isoforms [17,85]. In an

initial study, it was shown that, although PKCareduced p53-induced yeast growth inhibition ⁄ cell cyclearrest and PKCf had no effect on p53 activity, nPKCdand e enhanced p53 activity through its phosphoryla-

tion at Ser376-378 [17]. Similar results were obtained

in the presence of an apoptotic stimulus. In this case,

although cPKCa and aPKCf had no effect on p53

activity, nPKCd and e stimulated transcription-depen-

dent and -independent p53-mediated apoptosis [85]

(Fig. 4).

Indeed, important insights with respect to the mech-

anisms of apoptotic function of PKC isoforms, phar-

macological modulators and physiologically relevant

substrates have been provided by yeast PKC assays

(Table 1). Concerning PKCa, although frequently

described as an anti-apoptotic protein [61], contradic-

tory results suggesting a pro-apoptotic activity have

also been reported [88]. This pro-apoptotic function of

PKCa was corroborated in yeast. Indeed, this isoform

stimulated acetic acid-induced apoptosis, abolished the

Bcl-xL anti-apoptotic effect and stimulated the pro-

apoptotic activity of Bax. Despite this, p53 does not

appear to be an apoptotic substrate of PKCa(Table 1). Concerning PKCd, the well-known pro-

apoptotic function of this isoform in mammalian cells

[61,87] was widely confirmed in yeast. PKCd stimu-

lated acetic acid- and coleon U-induced apoptosis and

activated p53 (Table 1). Although several studies have

reported the phosphorylation of p53 and the activation

of its transcriptional mechanism by PKCd in mammals

[89–92], work in yeast has revealed for the first time

the involvement of this isoform in the regulation of a

p53 transcription-independent mechanism. Interest-

ingly, stimulation of H2O2-induced apoptosis by PKCdin yeast was only achieved in the presence of p53 [85].

This corroborated similar studies performed in mam-

malian cells, in which the stimulation of H2O2-induced

apoptosis was almost abolished in the presence of

rottlerin (a selective inhibitor of PKCd) and in null-

Fig. 4. Regulation of transcription-dependent and -independent p53 mechanisms by mammalian nPKCd and e in yeast. (A) Human wt p53

expressed in yeast presents nuclear localization and induces growth inhibition associated with S-phase cell cycle arrest, which is abolished

by the selective inhibitor of p53 transcriptional activity, PFT-a. Co-expression of mammalian nPKCd or e markedly increases the p53-induced

growth arrest through p53 phosphorylation [17,85]. (B) Under cell death conditions induced by H2O2 (cellular stress is highlighted with a yel-

low flash), nPKCd and e stimulate the translocation of a fraction of p53 from the nucleus to the mitochondria, and an increase in mitochon-

drial ROS production, mitochondrial transmembrane potential (DWm) loss and mitochondrial network fragmentation. This p53-mitochondrial

apoptotic pathway is markedly reduced by the absence of respiration (using strains devoid of mitochondrial DNA, rho0 mutants) and by the

selective inhibitor of mitochondrial p53 translocation, PFT-l [85].



p53 cells [89]. This may suggest that, for this stimulus,

the pro-apoptotic activity of PKCd is highly dependent

on a specific substrate that appears to be p53. It also

suggests that Bcl-xL is not a PKCd apoptotic substrate

(Table 1). Concerning PKCe, this isoform is frequently

regarded as having anti-apoptotic properties in mam-

malian cells [61,87]. Although the mechanisms respon-

sible for its anti-apoptotic function are still not well

clarified, it appears to involve the regulation of Bcl-2

family proteins [87], such as the inhibition of the pro-

apoptotic proteins Bax and Bad [93,94]. Indeed, the

yeast cell model revealed that PKCe also regulates

Bcl-xL anti-apoptotic activity [18].

Although several studies suggest that PKCe favours

life over death, other studies report the involvement of

this isoform in apoptosis promotion [95]. When

expressed in yeast, PKCe stimulated acetic acid-, co-

leon U- and H2O2-induced yeast apoptosis and p53

activity (Table 1). Taken together, the results obtained

in yeast suggest that the apoptotic function of PKCe is

highly dependent on its accessibility to key apoptotic

proteins, such as Bcl-xL and p53. The translocation of

PKCe to distinct subcellular compartments (e.g. as a

result of the distinct stimuli applied) may expose the

isoform to distinct substrates and this may be responsi-

ble for its distinct activities. Concerning PKCf, its

anti-apoptotic role in mammalian cell death signalling

pathways is also well-accepted [61]. However, it was

observed that PKCf stimulated acetic acid-induced

yeast apoptosis. Despite this, when this isoform was

co-expressed with Bcl-xL, it markedly enhanced

the Bcl-xL anti-apoptotic activity, with a complete

abolishment of acetic acid-induced apoptosis [18].

Interestingly, the yeast assay also suggests that p53 is

not an apoptotic substrate of PKCf (Table 1). Taken

together, although PKCa, d, e and f stimulated acetic

acid-induced apoptosis, only PKCe stimulated the

H2O2 apoptotic effect (Table 1). This indicates that,

for H2O2, the apoptotic substrates of PKCa, d and fare probably not conserved in yeast. These studies

emphasize that the apoptotic function of a specific

PKC isoform is highly dependent on the stimulus

applied.

Overall, valuable insights into the genetic, molecular

and functional profile of individual PKC isoforms have

been provided by the yeast PKC assays. Additionally,

potent and selective small molecule modulators of

these kinases have been identified. The studies dis-

cussed above certainly represent an important contri-

bution to the design of new therapeutic strategies

against cancer through the identification of key apop-

totic targets amongst the PKC family isoforms.

p53 tumour suppressor protein

The p53 tumour suppressor protein is a sequence-spe-

cific DNA-binding transcription factor that regulates

the expression of an assortment of genes involved in

cell cycle, apoptosis and numerous other processes.

Mutations in the TP53 gene are a feature of 50% of

all reported cancer cases. In the other cases where the

TP53 gene itself is not mutated, the p53 pathway is

often partially inactivated. This exemplifies the critical

role of p53 in human cancers. The regulation of the

p53 activity is therefore considered as a hallmark in

the fight against cancer. Hence, over the last 30 years,

p53 has become the focus of intensive basic and clini-

cal research [96].

The versatility of the yeast cell model for studying

several cellular processes, such as cell cycle and apop-

tosis, associated with the fact that no orthologues of

human p53 have been discovered so far in this

eukaryotic cell [97,98], has justified the attractiveness

of this cell system for studying p53. Indeed, despite the

complexity of p53 regulation in higher eukaryotes, at

the beginning of the 1990s, it was shown that human

wt p53 was also a sequence-dependent transcription

factor in yeast [99,100]. Additionally, it was shown

that p53 activating kinase (PAK1) was an essential

gene for the activation of p53 transcriptional activity

in yeast [101]. Subsquently, additional evidence was

provided corroborating the remarkable similarities

between the p53 transcriptional activity in yeast and

mammalian cells [98]. With this interesting discovery,

yeast became a powerful tool for uncovering major

aspects of the function of p53. For example, using

yeast, regulators of p53 transcriptional activity were

identified. It was shown that the p53 transcriptional

activity in yeast was subjected to redox regulation and

required thioredoxin reductase [102], as subsequently

confirmed in mammalian cells [103]. Moreover, Ishioka

Table 1. Effects of mammalian PKC isoforms on distinct apoptotic

stimuli and human apoptotic proteins expressed in yeast. (›),

Increase of effect; (fl), decrease of effect; (NE), no effect; (–), not

determined.

cPKCa nPKCd nPKCe aPKCf References

Apoptotic stimuli

Acetic acid › › › › [18]

H2O2 NE NE › NE [85]

Coleon U NE › › NE [79]

Apoptotic proteins

Bcl-xL fl NE › › [18]

Bax › – – – [84]

p53 NE › › NE [85]



et al. [104] developed the technique of functional anal-

ysis of separated alleles in yeast (FASAY) with the

aim of understanding the status of p53 in cancer cells.

Several modified versions of this FASAY assay were

subsequently used to identify tumour-derived p53 gene

mutations and to understand how these mutations

could interfere with the p53 function in human tumour

cells (Fig. 5). Indeed, the identification and classifica-

tion of tumour-derived p53 mutants based upon their

degree of loss of function has a high clinical value.

Yeast-based p53 functional assays were also revealed

to be a powerful tool for molecular epidemiology. The

assumption that carcinogens leave fingerprints sug-

gested that an analysis of the frequency, type and site

of mutations in genes frequently altered in carcinogen-

esis may provide clues with respect to the identification

of factors contributing to carcinogenesis [97,105].

Using these assays, it was demonstrated that p53

mutants had a partial loss of transcriptional activity

[106]. The ability and efficacy of p53 mutants to bind

and activate crucial reporters of cell cycle and apopto-

sis were also studied in yeast [107,108]. Moreover,

2314 p53 mutants, representing all the possible amino

acid substitutions caused by a point mutation, were

constructed, expressed and evaluated in yeast aiming

to understand the effect of all these mutations on p53

function [109]. In another study, yeast was used to

identify intragenic suppressor mutations that were able

to restore the activity to nonfunctional p53 mutants

[110].

Recently, an elegant yeast p53 transactivation assay

for the high-throughput screening of factors that can

influence p53 function, including mutations, cofactor

proteins and small molecules, was developed [111].

Using this assay, the interaction of p53 with its endog-

enous negative regulators, MDM2 and 53BP1, was

analyzed. The results obtained showed that MDM2

and 53BP1 led to a reduction in the p53 transactiva-

tion activity. However, unlike MDM2, which also

reduced the transactivation activity of p53 mutants

with a partial transactivation function, the impact of

53BP1 was lost or greatly reduced with specific partial

function p53 mutants. Concerning the ability of human

MDM2 to reduce p53 transactivation activity in yeast,

as initially reported in 1993 [112], it was also shown

that MDM2 can interact with endogenous yeast path-

ways to ubiquitylate and sumoylate p53. Ubiquityla-

tion led to p53 degradation, whereas sumoylation was

essential for the localization of p53–MDM2 complexes

to yeast nuclear bodies [113].

The induction of growth arrest by human wt p53

expressed either in S. cerevisiae [114] or S. pombe [115]

has also been highly explored in the study of p53. In

S. cerevisiae, it was reported that wt p53 caused a mild

growth inhibition [114,116], which was markedly

increased by the co-expression of human cell cycle-

regulated protein kinase [114]. Similarly, it was

also observed that the expression of human wt p53 in

S. cerevisiae induced growth inhibition associated with

S-phase cell cycle arrest, which was markedly increased

by PKCd and e. Additionally, using the selective p53

transcriptional inhibitor pifithrin (PFT)-a, a complete

abrogation of p53-induced growth arrest was obtained.

This supported the transcription of genes involved in

the yeast cell cycle by human p53 [79] (Fig. 4). Interest-

ingly, with the exploitation of this p53 phenotype in

S. pombe, it was shown that p53-induced cell cycle

arrest was abrogated by the cell cycle regulator protein

phosphatase cdc25 [115]. Subsequently, it was shown

Fig. 5. The p53 FASAY assay. It consists of the amplification of

p53 cDNA by RT-PCR (using p53 mRNA from tumour samples or

other tissues) and the co-transformation of a yeast reporter strain

with the PCR product and a gapped expression plasmid. (A) p53

status is evaluated using plates without histidine. Yeast clones

expressing a functional p53 (p53wt) are His+ as a result of a capac-

ity to express the HIS3 reporter gene (REP) and will therefore

grow; yeast clones expressing a mutated p53 cDNA (p53mt) are

His- and will not grow. (B) A modified version of the FASAY assay,

with ADE2 instead of HIS3 as a REP [122]. Using plates with low

concentration of adenine, p53 mutations are identified via the col-

our of the colonies. White colonies indicate a wt p53 that allowed

ADE2 expression and red colonies indicate a mutant p53. (Adapted

from [123]).



that the regulation of cell cycle progression by p53 in

mammalian cells involved its interaction with cdc25

[117]. More recently, Hadj Amor et al. [118] reported

that the expression of wt p53 in S. cerevisiae resulted in

apoptotic cell death. It was also revealed that p53 inter-

fered with the expression of the gene encoding the anti-

apoptotic and anti-oxidative protein, thioredoxin.

Because this protein has a crucial function in the pro-

tection of yeast cells against ROS, these results sug-

gested that p53 may induce apoptosis in part by down-

regulation of anti-apoptotic proteins. These different

phenotypes obtained with the expression of human p53

in yeast may be attributed to different p53 cellular lev-

els as a result of the use of different expression vectors.

Apoptosis induced by p53 is firmly established as a

central mechanism of tumour suppression [96]. In

1995, it was noted that the overexpression of a mutant

p53, lacking most of the DNA binding domain and

completely deficient in transactivation function, could

nonetheless trigger apoptosis [119]. That study was the

first reference concerning the capacity of p53 to induce

apoptosis independent of its transcriptional function.

At present, despite the prominence of p53 nuclear

transcriptional activity in the induction of apoptosis, a

transcription-independent mechanism involving mito-

chondrial p53 translocation is receiving increased

attention. Indeed, recent findings have provided

encouragement regarding further exploration of the

potential of mitochondrial p53-based cancer therapeu-

tics [96,120]. The first evidence for the conservation in

yeast of this transcription-independent p53-mediated

apoptosis was recently provided [85] (Fig. 4). It was

also shown that, besides the activation of p53 tran-

scriptional activity, nPKCd and e triggered the p53

translocation to mitochondria. This provided new

insights about the regulation of p53 translocation to

mitochondria. Additionally, the results obtained sup-

ported the possibility that mitochondrial p53 localiza-

tion is a subtle deciding factor that dictates whether

cells die or arrest growth. Indeed, mitochondrial p53

localization was only detected in yeast under an apop-

totic cell death scenario.

The direct participation of p53 in the intrinsic mito-

chondria-mediated apoptotic pathway may involve

interaction with the multidomain members of the Bcl-2

family, and particularly the activation of one of its

pro-apoptotic members Bax or Bak, to induce mito-

chondrial outer membrane permeabilization. However,

the underlying mechanisms remain to be completely

clarified. The recent discovery of Ybh3p in yeast [121]

may contribute to an understanding of how interac-

tions between p53 and Bcl-2 family members promote

mitochondrial outer membrane permeabilization.

Taken together, the studies referred to above show

that, although no p53 orthologues have been found in

yeast, the p53 pathway is highly conserved in this

eukaryotic organism. Additionally, several lines of evi-

dence are provided that highlight yeast as a powerful

cell model for use in genetic, molecular and functional

studies of p53.

Concluding remarks

Besides the recognized contribution of yeast to our

present understanding of many fundamental aspects of

cellular biology in higher eukaryotes, in recent years,

yeast has proved to be a powerful model organism for

unravelling the molecular basis of complex human dis-

eases such as cancer. Indeed, the ease of genetic

manipulation of yeast cells, in association with the

conservation of many features of higher eukaryotic

physiology, a tractable genome, a short generation

time and a large network of researchers who have gen-

erated a vast arsenal of experimental tools, has justi-

fied the extensive use of this model organism in

biomedicine.

In this review, the impact of using ‘humanized yeast

systems’ in the dissection of cancer-related molecular

processes and in the identification of genetic and phar-

macological regulators of this disease is discussed.

Studies involving the heterologous expression of pro-

teins in yeast are sometimes viewed with skepticism,

with toxicity often considered as a mere response to

unphysiological expression levels. Although it is unli-

kely that all of the protein functions revealed by yeast

will be relevant for human diseases, the numerous

insights into protein functions that are obtained from

yeast justifies the exploitation of this cell system.

More recently, sequencing of the human genome

promised the identification of new disease-causing

genes. This brought an enormous expectation for the

therapy of severe human disorders such as cancer.

However, this gave rise to the discovery of genes

whose normal function in these pathologies is com-

monly unknown. The genetic manipulations required

to uncover gene function are often extremely difficult

to perform in human cells. This lead to the ‘rebirth’ of

yeast as a valuable tool for uncovering the function of

human genes. We therefore anticipate that promising

new discoveries will continue in this area using yeast

as a model organism.

Acknowledgements

This work was supported by FCT (Fundacao para a

Ciencia e a Tecnologia) and FEDER funds through the



COMPETE program under the project FCOMP-01-

0124-FEDER-015752 (reference FCT PTDC ⁄SAU-

FAR ⁄110848 ⁄2009) and by the University of Porto ⁄Santander Totta. This work was also supported by FCT

through REQUIMTE (grant number PEst-C ⁄EQ-

B ⁄LA0006 ⁄2011) and through the fellowships of I. Cout-

inho (SFRH ⁄BD ⁄36066 ⁄2007), M. Leao (SFRH ⁄BD ⁄64184 ⁄2009), J. Soares (SFRH ⁄BD ⁄78971 ⁄2011) andC. Pereira (SFRH ⁄BPD ⁄44209 ⁄2008).

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New insights into cancer-related proteins provided by the yeast model

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