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RESEARCH ARTICLE
Functional characterization of dosage-dependent lethal
mutation of ubiquitin in Saccharomyces cerevisiae
Ankita Doshi, Pradeep Mishra, Mrinal Sharma & C. Ratna Prabha
Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India
Correspondence: C. Ratna Prabha,
Department of Biochemistry, Faculty of
Science, The Maharaja Sayajirao University of
Baroda, Vadodara 390002, India.
Tel.: +91 265 2795594;
fax: +91 265 2795569;
e-mail: [email protected]
Received 19 January 2014; revised 20 August
2014; accepted 25 August 2014. Final
version published online 2 October 2014.
DOI: 10.1111/1567-1364.12209
Editor: Monique Bolotin-Fukuhara
Keywords
ubiquitin; mutations in ubiquitin; in vitro
evolution of ubiquitin; structure of ubiquitin;
functions of ubiquitin.
Abstract
Ubiquitin is a eukaryotic protein with 96% sequence conservation from yeast
to human. Ubiquitin plays a central role in protein homeostasis and regulation
of protein function. We have reported on the generation of variants of ubiquitin by in vitro evolution in Saccharomyces cerevisiae to advance our understanding of the role of the invariant amino acid residues of ubiquitin in
relation to its function. One of the mutants generated, namely UbEP42, was a
dosage-dependent lethal form of the ubiquitin gene, causing lethality to UBI4deficient cells but not to ubiquitin wild-type cells. In the present study we
investigated the functional reasons for the observed lethality. Expression of
UbEP42 in a UBI4-deleted stress-sensitive strain resulted in an increased generation time due to a delayed S phase caused by decreased levels of Cdc28 protein kinase. Cells expressing UbEP42 displayed heightened sensitivity towards
heat stress and exposure to cycloheximide. Furthermore, its expression had a
negative effect on the degradation of substrates of the ubiquitin fusion degradation pathway. However, UbEP42 is incorporated into polyubiquitin chains.
Collectively, our results establish that the effects seen with the mutant ubiquitin
protein UbEP42 are not due to malfunction at the stage of polyubiquitination.
YEAST RESEARCH
Introduction
Ubiquitin is a eukaryotic protein employed as a tag in
the post-translational modification of numerous proteins
(Finley et al., 2012). Many key regulators of cell physiology such as cell cyclins (Pagano, 1997), transcription
factors (Hochstrasser & Varshavsky, 1990), tumor suppressors and DNA repair proteins (Jentsch et al., 1987)
are candidates for this post-translational modification,
known as ubiquitination (Varshavsky, 1997, 2012). Ubiquitination involves conjugation of the C-terminal carboxyl
of ubiquitin to the e-amino group of lysine residues of
the candidate protein (Hershko & Ciechanover, 1998).
Proteins modified through monoubiquitination have a
single molecule of ubiquitin attached to them. In other
cases, candidate proteins are polyubiquitinated as ubiquitin molecules are added to one another forming a polyubiquitin chain of four to five molecules on them
(Pickart & Fushman, 2004). Ubiquitin has seven lysine
residues and all of them are known to participate in polyubiquitin chain formation (Peng et al., 2003; Komander,
2009). The diversity in the linkages in polyubiquitin
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Published by John Wiley & Sons Ltd. All rights reserved
chains carry different functional meanings to candidate
proteins either by channeling them towards proteasomemediated degradation (Weissmann, 1997; Hershko &
Ciechanover, 1998; Komander, 2009) or regulating the
processes in which they participate, such as DNA repair
(Jentsch et al., 1987), chromatin dynamics (Levinger &
Varshavsky, 1982; Muratani & Tansey, 2003; Shilatifard,
2006) and lysosomal degradation in the case of
membrane proteins (Galan et al., 1996; Bonifacino &
Weissman, 1998; Nakatsu et al., 2000).
Ubiquitination is catalysed by a cascade of three
enzymes: ubiquitin activating enzyme or E1, ubiquitin
conjugating enzyme or E2 (Deshaies & Joazeiro, 2009; Varshavsky, 2012) and ubiquitin ligase or E3 (Jackson et al.,
2000; Deshaies & Joazeiro, 2009; Rotin & Kumar, 2009). A
sizable portion of the eukaryotic genome is dedicated to
encoding the enzymes of the ubiquitination pathway
(Finley et al., 2012). At the other end, deubiquitinating
enzymes act on the ubiquitinated proteins, freeing them
from the tag (Reyes-Turcu et al., 2009). In addition, there
are several receptor proteins that recognize the topology of
the polyubiquitin chain on candidate proteins.
FEMS Yeast Res 14 (2014) 1080–1089
1081
Studies on dosage dependent lethal ubiquitin mutant
Two significant aspects of ubiquitin biology are its
highly conserved protein sequence (Schlesinger & Goldstein, 1975; Gavilanes et al., 1982; Vierstra et al., 1986;
Wilkinson et al., 1986) and its universal presence in
eukaryotic cells. Sequence conservation of ubiquitin
ensures its interaction with the whole gamut of proteins
and enzymes that are integral to the ubiquitination pathway. Although knowledge of ubiquitination and its effects
on candidate proteins is growing, details of the importance of invariant residues, which are not part of interactive surfaces of ubiquitin, remain elusive. To understand
the contribution of these invariant amino acid residues to
structural and functional topology of the molecule we
have utilized in vitro evolution of the protein using errorprone PCR (Prabha et al., 2010). The resultant mutants
were screened in the SUB60 strain of Saccharomyces cerevisiae. In S. cerevisiae there are four genes encoding
ubiquitin, UBI1, UBI2, UBI3 and UBI4 (Ozkaynak et al.,
1984, 1987). The first three genes are required for normal
growth and survival of the organism, whereas UBI4 is
required for survival under stress conditions. Hence,
strain SUB60 lacking UBI4 is stress-hypersensitive. However, it can grow under conditions of stress, provided
ubiquitin is expressed extrachromosomally (Spence et al.,
1995). Alternatively, if a mutant form of ubiquitin is a
functional equivalent of wild-type ubiquitin, SUB60 cells
expressing the mutation would be able to overcome stress
conditions.
Following this logic, mutants generated by error-prone
PCR, in our previous study, were transformed into the
SUB60 strain of S. cerevisiae and screened for loss of tolerance towards temperature stress. Overexpression of one
of the mutations, namely UbEP42, showed the lethal phenotype even at permissive temperature. However, overexpression of the mutation did not have lethal effects on
SUB62 cells, which are wild-type for the UBI4 gene, leading to the conclusion that UbEP42 is a dosage-dependent
lethal mutation (Prabha et al., 2010). UbEP42 carries
amino acid substitutions in four positions, namely S20F,
A46S, L50P and I61T (Fig. 1 and Table 1). To understand
the possible reasons for the lethal phenotype observed,
β-turn
β-turn
MQ I F V K T L TG KT I T L EV E P S D T I E NV KAK I QDK E G I P P D Q
F
β-turn
β-turn
β-turn
QR L I F A GK Q L E D GR T L S D YN I QKE S T L HL V L R L R GG
S
P
T
Fig. 1. Amino acid sequence of ubiquitin along with its secondary
structure. The amino acid residue substitutions in UbEP42 are
indicated [adapted with permission from Prabha et al. (2010)].
FEMS Yeast Res 14 (2014) 1080–1089
functional aspects of ubiquitin –namely the influence of
the mutation on growth, sensitivity to cycloheximide,
degradation of proteins that are in-frame fusions of
ubiquitin either by the ubiquitin fusion degradation
(UFD) pathway or by the N-end rule pathway, were studied in SUB60 cells, in the background of UbEP42 expression. Finally, incorporation of UbEP42 into polyubiquitin
chains was investigated.
Materials and methods
Saccharomyces cerevisiae strains SUB62 (Mata, lys2-801
leu2-3,2-112 ura3-52 his3-Δ200 trp1-1) and SUB60 (Mata,
lys2-801, leu2-3,112, ura3-52, his3-Δ200, trp1-1, ubi4-Δ2::
LEU2) were used (Finley et al., 1987, 1994) for studying
the in vivo effects of UbEP42 protein.
Cultures were grown at 30 °C at 200 r.p.m. in synthetic dextrose (SD) medium containing 0.67% Hi-media
yeast nitrogen base (without amino acids) and 2% glucose as carbon source. Histidine (20 mg L1), lysine
(30 mg L1), uracil (20 mg L1), leucine (100 mg L1)
or tryptophan (20 mg L1) were added for selection,
depending on the experimental requirement (Finley et al.,
1994).
High copy number yeast episomal plasmid YEp96
(Finley et al., 1994) was used to express the genes for
wild-type ubiquitin and mutant ubiquitin in YEp96/
UbWt and YEp96/UbEP42, respectively. YEp96 is a shuttle vector between Escherichia coli and S. cerevisiae, with
TRP1 as a selection marker. The ubiquitin gene and its
variant UbEP42 were expressed from the CuSO4-inducible CUP1 promoter. Inducer concentration was standardized initially (see Supporting Information, Fig. S1)
and 100 lM CuSO4 was used in experiments where
induction was carried out for a shorter duration. In the
experiments where prolonged exposure to inducer was
necessary, 25 lM CuSO4 was used to avoid lethality
seen with the overexpression of UbEP42 at higher concentrations of CuSO4. To study the effect of UbEP42
expression on growth, the cultures of SUB60 transformed by YEp96/UbWt and YEp96/UbEP42 were grown
at 30 °C in SD media. Growth was monitored at
600 nm.
The cultures of SUB62 cells, SUB60 cells and transformants of SUB60 cells expressing UbWt and UbEP42
were grown to mid-log phase to approximately the
same OD600 nm. The cultures grown in three independent sets were then observed using confocal microscopy.
The images were captured using a Carl Zeiss laser scanning microscope, with 710 by 639 (oil immersion)
objective at a magnification of 6309 with DAPI filter.
Excitation and emission wavelengths were 403 and
430 nm, respectively.
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Published by John Wiley & Sons Ltd. All rights reserved
A. Doshi et al.
1082
Table 1. Amino acid substitutions in UbEP42, their location in the structure of ubiquitin, their hydropathy indices and secondary structural
preferences
Original residue and
its position
Substituting
residue
Structural feature of the protein
where substitution occurred
Kyte–Doolittle indices
for the original/substituting
residues
Secondary structural
preference for the original/
substituting residue
Ser20
Ala46
Leu50
Ile61
Phe
Ser
Pro
Thr
3rd residue of a Type I b turn
2nd residue of Type III b turn
b-sheet
Between two turns in a turn-rich region
0.8/2.8
1.8/0.8
3.8/1.6
4.5/0.7
1.06/0.96
0.96/1.23
1.30/0.55
–
The amino acid residues that have been substituted, the substitutions and secondary structural features affected by substitutions are given here.
The hydropathy scales of Kyte–Doolittle (1992) and original secondary structural preferences and substituting residues for b-turns as observed by
Hutchinson & Thornton (1994) and for b-sheets by Chou & Fasman (1978) are also indicated.
Protein extraction and Western blotting
experiments for estimating Cdc28 levels
Fresh cultures of SUB60, SUB60 transformed with
YEp96/UbWt and YEp96/UbEP42 were grown to log
phase (OD 0.6 at 600 nm) at 30 °C. Cells were harvested and washed twice with phosphate-buffered saline
(PBS), pH 8.0 (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HP04, 1.8 mM KH2PO4). The cells were then lysed
by sonication. Protein concentration was determined by
the Folin Lowry method. Fifty micrograms of protein
was loaded in each lane and was subjected to electrophoresis on 15% sodium dodecyl sulfate polyacrylamide
gels (SDS-PAGE). Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane
was then blocked by blocking buffer containing 5% (w/
v) nonfat dry milk in PBS-T for 1 h and the membrane
was probed by anti-Cdc28 antibody (Santa Cruz). The
membrane was than probed with goat antirabbit IgG
horseradish peroxidase conjugate (GeNei), and washed
with PBS-T six times and with PBS twice. A blot was
developed with the ECL Western Blotting Detection Kit
(Amersham Biosciences).
Sensitivity to heat stress
SUB62, SUB60 and the transformants of SUB60 by plasmids YEp96/UbWt and YEp96/UbEP42 were grown to
log phase until the optical density of the cultures reached
a value between 0.5 and 0.6. The cultures were plated on
SD selection media with 25 lM copper sulphate. Plates
were incubated at 40 °C for periods of 0, 4, 8, 12 and
16 h, shifted back to 30 °C and the colonies counted.
The experiment was repeated three times in independent
sets and the mean values are presented with error bars.
Sensitivity to cycloheximide exposure
Complementation potential of the ubiquitin variant
UbEP42 was tested using an antibiotic sensitivity test
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(Hanna et al., 2003). Untransformed controls SUB60,
SUB62 and SUB60 transformed with YEp96/UbWt,
YEp96/UbF45W and YEp96/UbEP42 were grown to log
phase until their optical density reached a value around
0.2. The cultures were then serially diluted threefold and
spotted on yeast-potato-dextrose media with cycloheximide (4 lg mL1) and copper sulphate inducer (25 lM).
Culture plates were incubated at 30 °C for 10 days.
Degradation of substrate ubiquitin fusions by
N-end rule and UFD pathways
SUB60 and SUB62 strains of S. cerevisiae and their
transformants with pUb23 were used to study the degradation of substrate ubiquitin fusions by the N-end
rule and UFD pathways. Plasmid pUB23 is a galactoseinducible shuttle vector expressing ubiquitin b-galactosidase fusion protein (Ub-X-bgal) with URA3 as selection
marker (Bachmair et al., 1986; Baker & Board, 1991).
Ub-X-bgal is a substrate used for studying degradation
of b-galactosidase following either the N-end rule pathway, where the first residue of b-galactosidase X is Met,
or by the UFD pathway, where X is Pro. The transformants of SUB60 carrying YEp96/UbWt and YEp96/
UbEP42 were also transformed by pUb23. UbiquitinX-b galactosidase (Ub-X-bgal) gene fusion is under
control of the galactose-inducible GAL10 promoter in
pUb23. To test the effects of UbEP42 expressed in background on the degradation of Ub-X-bgal by the N-end
rule (Johnson et al., 1992; Varshavsky, 1996) and UFD
pathways (Johnson et al., 1992, 1995), two variants of
the Ub-X-bgal gene fusion with X position as Met and
Pro were employed in independent sets, respectively.
The transformants were grown to mid-log phase at
30 °C, in synthetic galactose media to express Ub-Xbgal constitutively. CuSO4 was added at 100 lg mL1
to mid-log phase cultures to induce the expression of
UbWt and UbEP42 from YEp96/UbWt and YEp96/
UbEP42, respectively. The incubation was continued for
another 2 h. Cells were spun down and washed twice
FEMS Yeast Res 14 (2014) 1080–1089
1083
Studies on dosage dependent lethal ubiquitin mutant
with distilled water and resuspended in saline and the
OD600 nm was adjusted to 0.5. Protein concentration
was estimated by a modified Lowry method. b-Galactosidase was assayed to measure protein stability
using o-nitrophenyl thiogalactoside (ONPG) substrate
(Johnson et al., 1995; Varshavsky, 1996). The enzyme
assays were repeated in three independent sets and the
activity of b-galactosidase was measured in nanomoles
of ONPG converted per minute per milligram protein.
SEM values are given in the graph.
Western blot analysis of the levels of
Ub-Pro-bGal and Ub-Met-bgal
Fresh cultures of SUB60 transformed with YEp96/UbWt
and YEp96/UbEP42 were taken and cotransformed with
the two variants of pUb23, namely pUb23/Met and
pUb23/Pro. The vectors pUb23/Met and pUb23/Pro carry
genes for Ub-Met-b-galactosidase and Ub-Pro-b-galactosidase, respectively. SUB60 transformed with pUb23/Met
were used as a control. The cultures were grown to log
phase. CuSO4 was added to a final concentration of
100 lM to cultures and were allowed to grow for 2 h.
Cells were harvested and lysed by sonication. Samples
containing 50 lg protein were resolved on 7% SDS-PAGE.
Proteins were transferred to PVDF membrane following
the method described above. The PVDF membrane was
then probed with anti-b galactosidase antibody (fluorescein isothiocyanate; Novus Biologicals) overnight at 4 °C.
The membrane was than probed with goat antirabbit IgG
horseradish peroxidase conjugate (GeNei). Blotting was
developed by ECL (Amersham).
Western blot analysis of polyubiquitination
Plasmid pUb221 carries a chimeric gene for ubiquitin
(UbWt) with c-myc tag attached N-terminally. In
pUbEP42, the gene for wild-type ubiquitin was replaced
by the mutated form (UbEP42). Fresh cultures of SUB60,
SUB60 transformed with pUb221 (UbWt) and SUB60
transformed with pUbEP42 were inoculated and grown to
log phase at 30 °C. CuSO4 inducer was added to a final
concentration of 100 lM to cultures and the cultures
were grown overnight. Cells were harvested and lysed.
Protein extracts were resolved on 17% SDS-PAGE and
transferred to PVDF membrane following the method
described above. The membrane was probed with a
mouse monoclonal antibody conjugated to peroxidase,
namely anti-C-myc-Peroxidase (Roche), overnight at
4 °C. The Western blot was developed with a chromogenic substrate, 3,30 -diaminobenzidine, in the presence of
H2O2 and NiCl2 (Genei).
FEMS Yeast Res 14 (2014) 1080–1089
Results
Standardization of inducer concentration for
studying the in vivo effects of UbEP42
expression
A stress-hypersensitive SUB60 strain of S. cerevisiae,
which lacks the UBI4 gene, grows normally in the
absence of any kind of stress. Previous studies from our
laboratory established that over-expression of UbEP42 in
this strain caused cell lysis even under normal conditions.
SUB62, being a wild-type strain for UBI4, by contrast
remained unaffected by UbEP42 expression (Prabha
et al., 2010). These results suggested that UbEP42 seemed
to obstruct the process of protein degradation in SUB60
cells either by hampering the process of ubiquitination
or by blocking the recognition of polyubiquitin chains as
their integral component. This effect is seen only in
SUB60 cells as there is ubiquitin insufficiency, whereas in
SUB62 where ubiquitin is in ample supply, UbEP42 molecules are probably diluted out. To determine the validity
of our hypothesis, we investigated the consequences of
UbEP42 expression in SUB60 cells. To determine the
concentration of inducer that causes induction of sublethal levels of UbEP42 expression, cultures of SUB62,
SUB60, and SUB60 transformed with YEp96/UbWt and
YEp96/UbEP42 were grown in the presence of 0, 10, 25,
50, 75, 100, 150 and 200 lM concentrations of copper
sulphate. Copper sulphate at above 50 lM led to cell
lysis of strain SUB60 (data not shown). Based on this
observation, 25 lM copper sulphate was added in experiments where prolonged exposure to inducer was necessary. Copper sulphate at 100 lM was used in the
experiments where cells were incubated with inducer for
1–2 h.
Effects of UbEP42 expression on the growth
and generation time of S. cerevisiae
The effect of UbEP42 expression on the growth profile of
S. cerevisiae strain SUB60 was studied. SUB60 cells transformed with the mutant gene for UbEP42 showed
retarded growth with increased lag phase even in the
absence of an inducer CuSO4, when compared with
SUB60 transformed by plasmid YEp96/UbWt, carrying
the wild-type ubiquitin gene. The generation time of
SUB60 cells lacking UBI4 and SUB60 transformants
expressing the wild-type ubiquitin gene from plasmid was
2.5 and 2 h, respectively, which increased to 4 h in the
mutant UbEP42 in the absence of inducer CuSO4 and
8 h and above after inducing cells with 25 and 50 lM
CuSO4 respectively (Fig. 2).
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A. Doshi et al.
1084
12
UbWt
UbEP42
***
***
1
2
3
4
38
8
6
KDa
***
4
2
0
0 μM
25 μM
50 μM
Fig. 2. Comparison of the influence of extrachromosomal expression
of UbWt and UbEP42 on the generation time of Saccharomyces
cerevisiae strain SUB60. The concentration of copper sulphate was
varied at from 0, to 25 to 50 lM to vary the expression levels of
UbWt and UbEP42. ***P < 0.001.
Table 2. Confocal microscopic studies of cell cycle progression with
SUB62, SUB60, SUB60/UbWt and SUB60/UbEP42
Ubiquitin
variant
Percentage
cells in
G1 phase
Percentage
cells in
S phase
Percentage
cells in
G2 + M phases
SUB62
SUB60
SUB60/UbWt
SUB60/UbEP42
68.2
65.0
65.3
55.6
21.3
22.1
19.2
35.0
10.5
12.9
15.3
9.4
Confocal microscopy was used to reassess our results
following the growth profile. The mid-log phase cultures
of SUB62 cells, SUB60 cells and their transformants
expressing UbWt and UbEP42 were observed using confocal microscopy (Forsburg & Nurse, 1991) and the
results are presented in Table 2. (Representative confocal
microscopy images are presented in Fig. S2.) From these
results, it can be concluded that there is a delay in
S-phase leading to a reduction in growth rate. Hence, the
results obtained by confocal microscopy confirm our
observations made earlier. As Cdc28 is known to regulate
the transition of S. cerevisiae cells from G1 to S-phase
(Mendenhall et al., 1987; Wittenberg & Reed, 1988), the
level of Cdc28 was compared in SUB60 cells, and SUB60
cells expressing UbWt and UbEP42. From the results it is
clear that UbEP42 expression has a negative influence on
Cdc28 level (Fig. 3).
Complementation of stress-hypersensitive
phenotype by UbEP42
The ubiquitin genes UBI1, UBI2 and UBI3 maintain basal
levels of ubiquitin, which is required for normal functioning of cells. UBI4 supports the survival of cells under
stress conditions such as heat shock, starvation, UV damage, amino acid analogs and antibiotics. Therefore,
SUB60 yeast cells which lack the UBI4 polyubiquitin gene
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Published by John Wiley & Sons Ltd. All rights reserved
Fig. 3. Western blot analysis of Cdc28 protein kinase. Lane 1,
protein molecular weight marker; lane 2, SUB60 cells; lane 3, SUB60
cells transformed with Yep96/UbWt; lane 4, SUB60 cells transformed
with Yep96/UbEP42.
offer the right background for checking the complementation efficiency of mutant forms of ubiquitin under
stress conditions. SUB60 cells transformed with YEp96/
UbEP42 were exposed to heat stress and antibiotic stress
to test if UbEP42 could complement successfully and
rescue the cells under stress.
Heat stress complementation
The complementation ability of UbEP42 under heat stress
was tested by subjecting SUB60 cells expressing UbEP42
to heat stress at 40 °C for various intervals and returning
them back to permissive temperature (30 °C) to allow
normal growth. UbEP42 transformants of SUB60 cells fail
to revive after 4 h of incubation at 40 °C, showing more
severe effects than the untransformed SUB60 cells lacking
the UBI4 gene, while control SUB60 cells transformed
with the gene for UbWt were able to endure heat stress.
Thus, the result establishes the failure of UbEP42 in complementing SUB60 cells under heat stress (Fig. 4).
Antibiotic sensitivity test
Absence of the polyubiquitin gene UBI4 in SUB60 cells
makes them more sensitive to the antibiotic cycloheximide,
an effect nullified by expression of UbWt extrachromso-
100
Percentage survival (%)
Time (h)
10
SUB 60
SUB 62
UbWt
UbEP42
80
60
40
20
0
0
4
8
12
16
Incubation time (h)
Fig. 4. Effect of expression of UbEP42 on the survival of SUB60 cells
upon exposure to heat stress. SUB62, SUB60 and SUB60 transformed
by YEp96/UbWt and YEp96/UbEP42. The cells were exposed to heat
stress for different durations as shown in the graph and shifted to
30 °C.
FEMS Yeast Res 14 (2014) 1080–1089
1085
Studies on dosage dependent lethal ubiquitin mutant
1
2
3
4
1
2
3
4
SUB 62
SUB 60
SUB 60/YEp96/
UbWt
SUB 60/YEp96/
UbEP42
–CYCLOHEXIMIDE
+CYCLOHEXIMIDE
Fig. 5. Effect of expression of UbEP42 on Saccharomyces cerevisiae
(SUB60) under antibiotic stress. SUB60 cells transformed with YEp96/
UbEP42 were grown on plates containing cycloheximide. SUB62 cells
and SUB60 cells transformed with YEp96/UbWt were used as
controls. SUB60 and SUB60 transformed with UbEP42 fail to grow in
the presence of cycloheximide. Undiluted cultures (1), and threefold
serial dilutions (2–4) were spotted on YPD plates containing 25 lM
copper sulphate and 4 lg mL1 cycloheximide.
mally. To check if UbEP42 can complement in a similar
fashion, the complementation study was extended further
to cycloheximide stress. The two positive controls, SUB62
and SUB60 transformed with the gene for UbWt, could
resist the deleterious effect of cycloheximide. Expression of
the ubiquitin variant UbEP42 apparently renders SUB60
cells more sensitive to cycloheximide as compared with untransformed SUB60 cells lacking the UBI4 gene. The result
suggests that one or more of the amino acid residue substitutions occurring in UbEP42 make it functionally impaired
through structural changes (Fig. 5).
Specific acitivity (103)
65
FEMS Yeast Res 14 (2014) 1080–1089
Varshavsky’s group designed ubiquitin fusions of b-galactosidase to study protein degradation by the N-end
rule (Johnson et al., 1992; Varshavsky, 1996) and UFD
pathways (Johnson et al., 1992, 1995). They observed
that b-galactosidase fusions of ubiquitin with stabilizing
N-terminal residues such as Met (M), as in Ub-Metb-galactosidase, are cleaved by deubiquitinating enzyme
releasing a free Met-b-galactosidase which has a longer
half-life. b-Galactosidase fusions of ubiquitin with Pro at
their N terminus as in Ub-Pro-b-galactosidase are not
deubiquitinated. They therefore undergo polyubiquitination and subsequently head for degradation by the UFD
pathway. The ubiquitin-X-b-galactosidase fusion is under
GAL10 promoter control, where X is the N-terminal residue of b-galactosidase. Changes in the level of b-galactosidase activity can therefore reflect the effect of UbEP42 on
protein degradation. Thus, in the present study the two
substrates Ub-Met-b-galactosidase and Ub-Pro-b-galactosidase were chosen to understand the effect of UbEP42 on
protein degradation. The results indicate that Met-b-galactosidase activity remained more or less unchanged with
UbEP42 in the background (Fig. 6a). However, b-galactosidase activity increased with Pro-b-galactosidase in
SUB60 cells expressing UbEP42, as compared with those
transformed by genes for UbWt and UbF45W. Conversely,
(a)
45
25
5
–15
SUB60
SUB62
60/M
62/M
60/M/Wt
60/M/42
25
(b)
Specific acitivity (103)
Fig. 6. Effect of UbEP42 on the degradation
of proteins in the background lacking UbI4.
SUB60, SUB62, and SUB60 transformed by
plasmids YEp96/UbWt and YEp96/UbEP42
expressing the two forms of ubiquitin, namely
wild-type UbWt and UbEP42, were assayed for
b-galactosidase activity. These cells were also
transformed by pUb23/Met expressing UbMet-b-galactosidase fusion (a) and pUb23/Pro
expressing Ub-Pro-b-galactosidase fusion (b).
b-Galactosidase has Met (M) and Pro (P) as
the N-terminal residues. SUB60 and SUB62
used as controls in the experiment were also
transformed by plasmid pUb23/Met and
pUb23/Pro. ***P < 0.001.
Effect of the ubiquitin mutation on substrate
protein turnover by the N-end rule and UFD
pathways
***
20
15
10
5
0
–5
SUB60
SUB62
60/P
62/P
60/P/Wt
60/P/42
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Published by John Wiley & Sons Ltd. All rights reserved
A. Doshi et al.
1086
KDa
1
2
3
4
5
6
52
43
29
1
2
3
4
5
Fig. 7. Western blot analysis of b-galactosidase levels using anti-bgalactosidase antibody. SUB60 cells transformed with pUb23/Met and
Yep96/UbWt (lane 1), pUb23/Pro and Yep96/UbWt (lane 2), pUb23/
Met and Yep96/UbEP42 (lane 3), pUb23/Pro and Yep96/UbEP42 (lane
4), and SUB60 cells transformed with pUb23/Met alone as positive
control.
b-galactosidase activity of Ub-Pro-b-galactosidase in the
UbEP42 was is comparable to SUB60 expressing Ub-Prob-galactosidase alone (Fig. 6b). This again indicated loss
of function of UbEP42 in vivo. Hence, UbEP42 interferes
with the operation of the UFD pathway in S. cerevisiae.
Western blot analysis using anti-b-galactosidase antibodies confirmed the results on protein stability obtained
with b-galactosidase assays (Fig. 7).
Incorporation of UbEP42 into polyubiquitin
chains
High levels of Pro-b-galactosidase activity in SUB60 with
UbEP42 expression indicated two possibilities: either
UbEP42 is not recognized by the ubiquitination system or
it is recognized by components of the ubiquitination system and incorporated into polyubiquitin chains, whereby
it acts as an inhibitor for protein degradation. Wild-type
ubiquitin (UbWt) tagged with c-myc was shown to be conjugated to candidate proteins and extended into polyubiquitin chains, in a processing step required for the
specific degradation of candidate proteins (Ellison &
Hochstrasser, 1991). In the present experiment, UbEP42
was similarly tagged with c-myc to follow its incorporation
into polyubiquitin chains in SUB60. c-myc-tagged UbWt
was used as a positive control.
Immunoblot analysis of c-myc-tagged UbEP42 indicated that UbEP42 is incorporated into polyubiquitin
chains, suggesting that the conjugation is not affected by
mutations (Fig. 8). Hence, the function that could be
affected is the selective and regulated degradation of proteins. This result also explains the lethal effect of UbEP42
overexpression. The overexpression of UbEP42 far exceeds
the basal levels of wild-type ubiquitin in SUB60. Proteins
ubiquitinated with UbEP42 probably accumulate in the
system, causing lethality.
Together our results show that UbEP42 can be used
normally by the cell for polyubiquitination. However,
UbEP42 failed to complement SUB60 cells under heat
stress and antibiotic stress. It was observed that the
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
20.1
14.3
Fig. 8. Western blot showing ubiquitination profile. Lane 1, molecular
weight marker. Position of bands and their molecular weights are
given for comparison. Lanes 2 and 4, uninduced SUB60 cells carrying
pUb221 and pUbEP42, respectively; lanes 3 and 5, SUB60 cells
carrying pUb221 and pUbEP42, respectively, in the presence of
100 lM inducer; lane 6, untransformed SUB60 as negative control.
Both UbWt and UbEP42 have N-terminal myc-tag and anti-myc
antibodies were used for Western blotting. Ubiquitin in SUB60 cells in
lane 6 does not carry the tag and so no bands are seen, while a
characteristic polyubiquitination ladder is seen in lanes 3 and 5.
UbEP42 can therefore be incorporated into polyubiquitin chains.
mutant has a negative effect on the degradation of
Ub-Pro-b-galactosidase, implying that the polyubiquitin
chain of UbEP42 is not recognized by proteasomes for
degradation. The protein also could not rescue the
cells from heat stress, probably due to its thermal
instability.
Discussion
Ubiquitin serves as an example of a protein that strikes
an optimal balance between structure and function early
on evolution, undergoing just three changes in sequence
from yeast to human (Mishra et al., 2009). Ubiquitin
interacts with innumerable enzymes in the process of
ubiquitination and deubiquitination of a variety of candidate proteins belonging to diverse pathways. With the
benefit of hindsight, it appears that ubiquitin’s chances of
accumulating mutations were obliterated to avoid coevolution of interactive partners to maintain their interactions with an evolving ubiquitin. However, this feat of
conservation did not leave any room for understanding
the functional role of individual residues in ubiquitin.
Sloper-Mould et al. (2001) observed that there are
three functionally important surfaces in ubiquitin. They
also established that Ser20Ala and Ala46Gly substitutions
do not have any significant influence over growth, temperature and cold sensitivity of S. cerevisiae. Besides,
studies on ubiquitin binding domains (UBDs) pointed
to two types of interactions between ubiquitin and
UBDs, which involved other residues (Harper & SchulFEMS Yeast Res 14 (2014) 1080–1089
1087
Studies on dosage dependent lethal ubiquitin mutant
man, 2006; Dikic et al., 2009; Komander, 2009; Bomar
et al., 2010).
Previously, our laboratory has focused on the parallel
b-bulge of ubiquitin to gain insight into its structure–
function relationships (Mishra et al., 2009, 2011; Sharma
& Prabha, 2011; Prabha et al., 2012). The residues in the
b-bulge were substituted using site-directed mutagenesis.
The amino acid residue replacements were chosen to preserve the structure of ubiquitin intact and the functional
integrity of the resultant variants was studied. Although
this approach is highly specific, it is limited only to the
site of choice and the residue chosen for replacement. On
the other hand, generation of mutants using in vitro evolution gives rise to a library of mutations. Selection and
characterization of individual mutations generated using
error-prone PCR in such cases can expand our knowledge
more rapidly.
In the present study, we have characterized the dosage-dependent ubiquitin lethal mutant UbEP42. The
mutant of ubiquitin UbEP42 has four mutations, which
render the protein thermally unstable. The hydropathy
indices and secondary structural preferences of the original and substituting residues are widely different in all
four cases (Table 1). Low level expression of UbEP42 in
SUB60 cells with UBI4 deletion did not help them withstand exposure to cycloheximide. The presence of
UbEP42 in the cellular ubiquitin pool impedes the degradation of the substrates of the UFD pathway. Incorporation of UbEP42 into polyubiquitin chains rules out
deficiency of ubiquitin as a likely cause for lethality,
unlike the case with strain SUB60. Incorporation of a
functionally inept ubiquitin leading to failure of degradation of proteins is a likely reason for the lethality
observed in this case.
In UbEP42 the substitutions of Ser20 to Phe and
Ala46 to Ser occurring in type I and type III turns,
respectively (Vijay-kumar et al., 1987), have similar propensities for the secondary structures. Furthermore,
despite large differences in their hydropathy indices,
these substitutions could be accommodated due to their
location on the surface of the protein, as suggested from
our more recent observations (M. Sharma, A. Doshi and
C.R. Prabha, unpublished observations). Moreover, the
e-amino group of Lys48 is known to be directly engaged
in H-bond with Ala46. The third substitution of Leu50
to Pro occurring in the last residue of the b-strand can
have a considerable negative impact on the structure
and stability of the protein as Pro has very low propensity for the b-strand. Furthermore, both Ala46Ser and
Leu50Pro occur closer to the two functionally important
residues Ile44 and Lys48. The substitution of Ile61 by
Thr affects one of the residues protected early during
the refolding of ubiquitin (Briggs & Roder, 1992). The
FEMS Yeast Res 14 (2014) 1080–1089
side chain of Ile61 is also buried in a hydrophobic
pocket formed by Ala46 and Leu67 of the wild-type
protein and hence the replacement hydrophobic residues
by polar residues in Ala46Ser and Ile41Thr are meant to
complement each other. The structure of ubiquitin is
stabilized by its globular shape with a hydrophobic core
and extensive hydrogen bonding. These mutations successfully explain its functional impairments and the
observed decrease in thermal stability of UbEP42. The
mutations described here, Ser20Phe and Ala46Ser, are
different from those studied by Sloper-Mould et al.
(2001) and the residues Leu50 and Ile61 did not form
part of the set of mutations chosen by them.
Although we wanted to study the role of individual residues in ubiquitin using single mutants, the UbEP42
mutant generated by error-prone PCR happened to carry
four amino acid substitutions (Prabha et al., 2010). Characterization of individual mutations of UbEP42 and their
combinations may reveal two important aspects of these
mutations. First, they highlight the functional contributions made by these four residues to ubiquitin biology.
Secondly, they unravel any cumulative or compensatory
influences the mutations are exerting over each other in
UbEP42. Third and most importantly, expressing UbEP42
in higher organisms under tissue-specific promoters may
lead to targeted removal of proteins where derailed protein degradation is the underlying cause for pathogenesis.
In conclusion, a dosage-dependent lethal mutation of
ubiquitin UbEP42, despite being incorporated into polyubiquitin chains, does not compensate for the deficiency
of UBI4 in S. cerevisiae strain SUB60, leaving it susceptible to heat stress and antibiotic stress. Under normal conditions overexpression of UbEP42 in UBI4-deleted strains
caused cell lysis, while low levels led to a competitive
inhibitor-like effect that slowed growth of the organism
considerably. Furthermore, UbEP42 has reduced the level
of Cdc28 protein kinase. Besides, the UbEP42 background
hampers the degradation of chimeric fusions of ubiquitin
degraded by the UFD pathway. Harnessing these observations made with UbEP42 may assist the development of
novel approaches in the treatment malignancies and neurodegenerative disorders.
Acknowledgements
C.R.P. is grateful to the University Grants Commission
for the Major Research Project [No. F.33-225/2007 (SR)].
C.R.P. thanks Professor Mark Searle and Professor Daniel
Finley for providing the plasmids and strains necessary
for the study. We thank the DBT-ILSPARE facility of The
M. S. University of Baroda, India, for use of the confocal
microscopy facilities. The authors have no conflict of
interest to declare.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
1088
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Modulation of UbEP42 expression levels by varying inducer copper sulphate concentration.
Fig. S2. Confocal microscopic pictures of SUB62, SUB60
strains of Saccharomyces cerevisiae and SUB60 cells
expressing UbEP42 from plasmid.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved