Functional characterization of dosage-dependent lethal mutation of ubiquitin in Saccharomyces cerevisiae


                 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

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                 Correspondence: C. Ratna Prabha,                       Abstract
                 Department of Biochemistry, Faculty of
                 Science, The Maharaja Sayajirao University of          Ubiquitin is a eukaryotic protein with 96% sequence conservation from yeast
                 Baroda, Vadodara 390002, India.                        to human. Ubiquitin plays a central role in protein homeostasis and regulation
                 Tel.: +91 265 2795594;                                 of protein function. We have reported on the generation of variants of ubiqu-
                 fax: +91 265 2795569;                                  itin by in vitro evolution in Saccharomyces cerevisiae to advance our under-
                                                                        standing 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
                 Received 19 January 2014; revised 20 August
                 2014; accepted 25 August 2014. Final
                                                                        dosage-dependent lethal form of the ubiquitin gene, causing lethality to UBI4-
                 version published online 2 October 2014.               deficient cells but not to ubiquitin wild-type cells. In the present study we
                                                                        investigated the functional reasons for the observed lethality. Expression of
                 DOI: 10.1111/1567-1364.12209                           UbEP42 in a UBI4-deleted stress-sensitive strain resulted in an increased gener-
                                                                        ation time due to a delayed S phase caused by decreased levels of Cdc28 pro-
                 Editor: Monique Bolotin-Fukuhara                       tein 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 degra-
                 ubiquitin; mutations in ubiquitin; in vitro
                                                                        dation pathway. However, UbEP42 is incorporated into polyubiquitin chains.
                 evolution of ubiquitin; structure of ubiquitin;
                 functions of ubiquitin.                                Collectively, our results establish that the effects seen with the mutant ubiquitin
                                                                        protein UbEP42 are not due to malfunction at the stage of polyubiquitination.

                                                                                              chains carry different functional meanings to candidate
                                                                                              proteins either by channeling them towards proteasome-
                 Ubiquitin is a eukaryotic protein employed as a tag in                       mediated degradation (Weissmann, 1997; Hershko &
                 the post-translational modification of numerous proteins                     Ciechanover, 1998; Komander, 2009) or regulating the
                 (Finley et al., 2012). Many key regulators of cell physiol-                  processes in which they participate, such as DNA repair

                 ogy such as cell cyclins (Pagano, 1997), transcription                       (Jentsch et al., 1987), chromatin dynamics (Levinger &
                 factors (Hochstrasser & Varshavsky, 1990), tumor sup-                        Varshavsky, 1982; Muratani & Tansey, 2003; Shilatifard,
                 pressors and DNA repair proteins (Jentsch et al., 1987)                      2006) and lysosomal degradation in the case of
                 are candidates for this post-translational modification,                     membrane proteins (Galan et al., 1996; Bonifacino &
                 known as ubiquitination (Varshavsky, 1997, 2012). Ubiq-                      Weissman, 1998; Nakatsu et al., 2000).
                 uitination involves conjugation of the C-terminal carboxyl                      Ubiquitination is catalysed by a cascade of three
                 of ubiquitin to the e-amino group of lysine residues of                      enzymes: ubiquitin activating enzyme or E1, ubiquitin
                 the candidate protein (Hershko & Ciechanover, 1998).                         conjugating enzyme or E2 (Deshaies & Joazeiro, 2009; Var-
                 Proteins modified through monoubiquitination have a                          shavsky, 2012) and ubiquitin ligase or E3 (Jackson et al.,
                 single molecule of ubiquitin attached to them. In other                      2000; Deshaies & Joazeiro, 2009; Rotin & Kumar, 2009). A
                 cases, candidate proteins are polyubiquitinated as ubiqu-                    sizable portion of the eukaryotic genome is dedicated to
                 itin molecules are added to one another forming a poly-                      encoding the enzymes of the ubiquitination pathway
                 ubiquitin chain of four to five molecules on them                            (Finley et al., 2012). At the other end, deubiquitinating
                 (Pickart & Fushman, 2004). Ubiquitin has seven lysine                        enzymes act on the ubiquitinated proteins, freeing them
                 residues and all of them are known to participate in poly-                   from the tag (Reyes-Turcu et al., 2009). In addition, there
                 ubiquitin chain formation (Peng et al., 2003; Komander,                      are several receptor proteins that recognize the topology of
                 2009). The diversity in the linkages in polyubiquitin                        the polyubiquitin chain on candidate proteins.

                 ª 2014 Federation of European Microbiological Societies.                                                       FEMS Yeast Res 14 (2014) 1080–1089
                 Published by John Wiley & Sons Ltd. All rights reserved
Studies on dosage dependent lethal ubiquitin mutant                                                                                         1081

   Two significant aspects of ubiquitin biology are its                  functional aspects of ubiquitin –namely the influence of
highly conserved protein sequence (Schlesinger & Gold-                   the mutation on growth, sensitivity to cycloheximide,
stein, 1975; Gavilanes et al., 1982; Vierstra et al., 1986;              degradation of proteins that are in-frame fusions of
Wilkinson et al., 1986) and its universal presence in                    ubiquitin either by the ubiquitin fusion degradation
eukaryotic cells. Sequence conservation of ubiquitin                     (UFD) pathway or by the N-end rule pathway, were stud-
ensures its interaction with the whole gamut of proteins                 ied in SUB60 cells, in the background of UbEP42 expres-
and enzymes that are integral to the ubiquitination path-                sion. Finally, incorporation of UbEP42 into polyubiquitin
way. Although knowledge of ubiquitination and its effects                chains was investigated.
on candidate proteins is growing, details of the impor-
tance of invariant residues, which are not part of interac-
                                                                         Materials and methods
tive surfaces of ubiquitin, remain elusive. To understand

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the contribution of these invariant amino acid residues to               Saccharomyces cerevisiae strains SUB62 (Mata, lys2-801
structural and functional topology of the molecule we                    leu2-3,2-112 ura3-52 his3-Δ200 trp1-1) and SUB60 (Mata,
have utilized in vitro evolution of the protein using error-             lys2-801, leu2-3,112, ura3-52, his3-Δ200, trp1-1, ubi4-Δ2::
prone PCR (Prabha et al., 2010). The resultant mutants                   LEU2) were used (Finley et al., 1987, 1994) for studying
were screened in the SUB60 strain of Saccharomyces cere-                 the in vivo effects of UbEP42 protein.
visiae. In S. cerevisiae there are four genes encoding                      Cultures were grown at 30 °C at 200 r.p.m. in syn-
ubiquitin, UBI1, UBI2, UBI3 and UBI4 (Ozkaynak et al.,                   thetic dextrose (SD) medium containing 0.67% Hi-media
1984, 1987). The first three genes are required for normal               yeast nitrogen base (without amino acids) and 2% glu-
growth and survival of the organism, whereas UBI4 is                     cose as carbon source. Histidine (20 mg L1), lysine
required for survival under stress conditions. Hence,                    (30 mg L1), uracil (20 mg L1), leucine (100 mg L1)
strain SUB60 lacking UBI4 is stress-hypersensitive. How-                 or tryptophan (20 mg L1) were added for selection,
ever, it can grow under conditions of stress, provided                   depending on the experimental requirement (Finley et al.,
ubiquitin is expressed extrachromosomally (Spence et al.,                1994).
1995). Alternatively, if a mutant form of ubiquitin is a                    High copy number yeast episomal plasmid YEp96
functional equivalent of wild-type ubiquitin, SUB60 cells                (Finley et al., 1994) was used to express the genes for
expressing the mutation would be able to overcome stress                 wild-type ubiquitin and mutant ubiquitin in YEp96/
conditions.                                                              UbWt and YEp96/UbEP42, respectively. YEp96 is a shut-
   Following this logic, mutants generated by error-prone                tle vector between Escherichia coli and S. cerevisiae, with
PCR, in our previous study, were transformed into the                    TRP1 as a selection marker. The ubiquitin gene and its
SUB60 strain of S. cerevisiae and screened for loss of tol-              variant UbEP42 were expressed from the CuSO4-induc-
erance towards temperature stress. Overexpression of one                 ible CUP1 promoter. Inducer concentration was stan-
of the mutations, namely UbEP42, showed the lethal phe-                  dardized initially (see Supporting Information, Fig. S1)
notype even at permissive temperature. However, over-                    and 100 lM CuSO4 was used in experiments where
expression of the mutation did not have lethal effects on                induction was carried out for a shorter duration. In the
SUB62 cells, which are wild-type for the UBI4 gene, lead-                experiments where prolonged exposure to inducer was
ing to the conclusion that UbEP42 is a dosage-dependent                  necessary, 25 lM CuSO4 was used to avoid lethality
lethal mutation (Prabha et al., 2010). UbEP42 carries                    seen with the overexpression of UbEP42 at higher con-
amino acid substitutions in four positions, namely S20F,                 centrations of CuSO4. To study the effect of UbEP42
A46S, L50P and I61T (Fig. 1 and Table 1). To understand                  expression on growth, the cultures of SUB60 trans-
the possible reasons for the lethal phenotype observed,                  formed by YEp96/UbWt and YEp96/UbEP42 were grown
                                                                         at 30 °C in SD media. Growth was monitored at
                                                                         600 nm.
             β-turn            β-turn                                       The cultures of SUB62 cells, SUB60 cells and trans-
                                                                         formants of SUB60 cells expressing UbWt and UbEP42
                                                                         were grown to mid-log phase to approximately the
                                                                         same OD600 nm. The cultures grown in three indepen-
        β-turn        β-turn                 β-turn
                                                                         dent sets were then observed using confocal microscopy.
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          The images were captured using a Carl Zeiss laser scan-
         S        P                      T
                                                                         ning microscope, with 710 by 639 (oil immersion)
Fig. 1. Amino acid sequence of ubiquitin along with its secondary        objective at a magnification of 6309 with DAPI filter.
structure. The amino acid residue substitutions in UbEP42 are            Excitation and emission wavelengths were 403 and
indicated [adapted with permission from Prabha et al. (2010)].           430 nm, respectively.

FEMS Yeast Res 14 (2014) 1080–1089                                                        ª 2014 Federation of European Microbiological Societies.
                                                                                            Published by John Wiley & Sons Ltd. All rights reserved
1082                                                                                                                                      A. Doshi et al.

Table 1. Amino acid substitutions in UbEP42, their location in the structure of ubiquitin, their hydropathy indices and secondary structural

                                                                                            Kyte–Doolittle indices           Secondary structural
Original residue and        Substituting       Structural feature of the protein            for the original/substituting    preference for the original/
its position                residue            where substitution occurred                  residues                         substituting residue
Ser20                       Phe                3rd residue of a Type I b turn               0.8/2.8                         1.06/0.96
Ala46                       Ser                2nd residue of Type III b turn                1.8/0.8                        0.96/1.23
Leu50                       Pro                b-sheet                                       3.8/1.6                        1.30/0.55
Ile61                       Thr                Between two turns in a turn-rich region       4.5/0.7                        –
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.

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                                                                                   (Hanna et al., 2003). Untransformed controls SUB60,
Protein extraction and Western blotting
                                                                                   SUB62 and SUB60 transformed with YEp96/UbWt,
experiments for estimating Cdc28 levels
                                                                                   YEp96/UbF45W and YEp96/UbEP42 were grown to log
Fresh cultures of SUB60, SUB60 transformed with                                    phase until their optical density reached a value around
YEp96/UbWt and YEp96/UbEP42 were grown to log                                      0.2. The cultures were then serially diluted threefold and
phase (OD 0.6 at 600 nm) at 30 °C. Cells were har-                                 spotted on yeast-potato-dextrose media with cyclohexi-
vested and washed twice with phosphate-buffered saline                             mide (4 lg mL1) and copper sulphate inducer (25 lM).
(PBS), pH 8.0 (137 mM NaCl, 2.7 mM KCl, 10 mM                                      Culture plates were incubated at 30 °C for 10 days.
Na2HP04, 1.8 mM KH2PO4). The cells were then lysed
by sonication. Protein concentration was determined by
                                                                                   Degradation of substrate ubiquitin fusions by
the Folin Lowry method. Fifty micrograms of protein
                                                                                   N-end rule and UFD pathways
was loaded in each lane and was subjected to electro-
phoresis on 15% sodium dodecyl sulfate polyacrylamide                              SUB60 and SUB62 strains of S. cerevisiae and their
gels (SDS-PAGE). Proteins were transferred to a polyv-                             transformants with pUb23 were used to study the deg-
inylidene fluoride (PVDF) membrane. The membrane                                   radation of substrate ubiquitin fusions by the N-end
was then blocked by blocking buffer containing 5% (w/                              rule and UFD pathways. Plasmid pUB23 is a galactose-
v) nonfat dry milk in PBS-T for 1 h and the membrane                               inducible shuttle vector expressing ubiquitin b-galactosi-
was probed by anti-Cdc28 antibody (Santa Cruz). The                                dase fusion protein (Ub-X-bgal) with URA3 as selection
membrane was than probed with goat antirabbit IgG                                  marker (Bachmair et al., 1986; Baker & Board, 1991).
horseradish peroxidase conjugate (GeNei), and washed                               Ub-X-bgal is a substrate used for studying degradation
with PBS-T six times and with PBS twice. A blot was                                of b-galactosidase following either the N-end rule path-
developed with the ECL Western Blotting Detection Kit                              way, where the first residue of b-galactosidase X is Met,
(Amersham Biosciences).                                                            or by the UFD pathway, where X is Pro. The transfor-
                                                                                   mants of SUB60 carrying YEp96/UbWt and YEp96/
                                                                                   UbEP42 were also transformed by pUb23. Ubiquitin-
Sensitivity to heat stress
                                                                                   X-b galactosidase (Ub-X-bgal) gene fusion is under
SUB62, SUB60 and the transformants of SUB60 by plas-                               control of the galactose-inducible GAL10 promoter in
mids YEp96/UbWt and YEp96/UbEP42 were grown to                                     pUb23. To test the effects of UbEP42 expressed in back-
log phase until the optical density of the cultures reached                        ground on the degradation of Ub-X-bgal by the N-end
a value between 0.5 and 0.6. The cultures were plated on                           rule (Johnson et al., 1992; Varshavsky, 1996) and UFD
SD selection media with 25 lM copper sulphate. Plates                              pathways (Johnson et al., 1992, 1995), two variants of
were incubated at 40 °C for periods of 0, 4, 8, 12 and                             the Ub-X-bgal gene fusion with X position as Met and
16 h, shifted back to 30 °C and the colonies counted.                              Pro were employed in independent sets, respectively.
The experiment was repeated three times in independent                             The transformants were grown to mid-log phase at
sets and the mean values are presented with error bars.                            30 °C, in synthetic galactose media to express Ub-X-
                                                                                   bgal constitutively. CuSO4 was added at 100 lg mL1
                                                                                   to mid-log phase cultures to induce the expression of
Sensitivity to cycloheximide exposure
                                                                                   UbWt and UbEP42 from YEp96/UbWt and YEp96/
Complementation potential of the ubiquitin variant                                 UbEP42, respectively. The incubation was continued for
UbEP42 was tested using an antibiotic sensitivity test                             another 2 h. Cells were spun down and washed twice

ª 2014 Federation of European Microbiological Societies.                                                               FEMS Yeast Res 14 (2014) 1080–1089
Published by John Wiley & Sons Ltd. All rights reserved
Studies on dosage dependent lethal ubiquitin mutant                                                                              1083

with distilled water and resuspended in saline and the
OD600 nm was adjusted to 0.5. Protein concentration           Results
was estimated by a modified Lowry method. b-Galacto-
                                                              Standardization of inducer concentration for
sidase was assayed to measure protein stability
                                                              studying the in vivo effects of UbEP42
using o-nitrophenyl thiogalactoside (ONPG) substrate
(Johnson et al., 1995; Varshavsky, 1996). The enzyme
assays were repeated in three independent sets and the        A stress-hypersensitive SUB60 strain of S. cerevisiae,
activity of b-galactosidase was measured in nanomoles         which lacks the UBI4 gene, grows normally in the
of ONPG converted per minute per milligram protein.           absence of any kind of stress. Previous studies from our
SEM values are given in the graph.                            laboratory established that over-expression of UbEP42 in
                                                              this strain caused cell lysis even under normal conditions.

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                                                              SUB62, being a wild-type strain for UBI4, by contrast
Western blot analysis of the levels of
Ub-Pro-bGal and Ub-Met-bgal
                                                              remained unaffected by UbEP42 expression (Prabha
                                                              et al., 2010). These results suggested that UbEP42 seemed
Fresh cultures of SUB60 transformed with YEp96/UbWt           to obstruct the process of protein degradation in SUB60
and YEp96/UbEP42 were taken and cotransformed with            cells either by hampering the process of ubiquitination
the two variants of pUb23, namely pUb23/Met and               or by blocking the recognition of polyubiquitin chains as
pUb23/Pro. The vectors pUb23/Met and pUb23/Pro carry          their integral component. This effect is seen only in
genes for Ub-Met-b-galactosidase and Ub-Pro-b-galactosi-      SUB60 cells as there is ubiquitin insufficiency, whereas in
dase, respectively. SUB60 transformed with pUb23/Met          SUB62 where ubiquitin is in ample supply, UbEP42 mol-
were used as a control. The cultures were grown to log        ecules are probably diluted out. To determine the validity
phase. CuSO4 was added to a final concentration of            of our hypothesis, we investigated the consequences of
100 lM to cultures and were allowed to grow for 2 h.          UbEP42 expression in SUB60 cells. To determine the
Cells were harvested and lysed by sonication. Samples         concentration of inducer that causes induction of suble-
containing 50 lg protein were resolved on 7% SDS-PAGE.        thal levels of UbEP42 expression, cultures of SUB62,
Proteins were transferred to PVDF membrane following          SUB60, and SUB60 transformed with YEp96/UbWt and
the method described above. The PVDF membrane was             YEp96/UbEP42 were grown in the presence of 0, 10, 25,
then probed with anti-b galactosidase antibody (fluores-      50, 75, 100, 150 and 200 lM concentrations of copper
cein isothiocyanate; Novus Biologicals) overnight at 4 °C.    sulphate. Copper sulphate at above 50 lM led to cell
The membrane was than probed with goat antirabbit IgG         lysis of strain SUB60 (data not shown). Based on this
horseradish peroxidase conjugate (GeNei). Blotting was        observation, 25 lM copper sulphate was added in experi-
developed by ECL (Amersham).                                  ments where prolonged exposure to inducer was neces-
                                                              sary. Copper sulphate at 100 lM was used in the
                                                              experiments where cells were incubated with inducer for
Western blot analysis of polyubiquitination
                                                              1–2 h.
Plasmid pUb221 carries a chimeric gene for ubiquitin
(UbWt) with c-myc tag attached N-terminally. In
                                                              Effects of UbEP42 expression on the growth
pUbEP42, the gene for wild-type ubiquitin was replaced
                                                              and generation time of S. cerevisiae
by the mutated form (UbEP42). Fresh cultures of SUB60,
SUB60 transformed with pUb221 (UbWt) and SUB60                The effect of UbEP42 expression on the growth profile of
transformed with pUbEP42 were inoculated and grown to         S. cerevisiae strain SUB60 was studied. SUB60 cells trans-
log phase at 30 °C. CuSO4 inducer was added to a final        formed with the mutant gene for UbEP42 showed
concentration of 100 lM to cultures and the cultures          retarded growth with increased lag phase even in the
were grown overnight. Cells were harvested and lysed.         absence of an inducer CuSO4, when compared with
Protein extracts were resolved on 17% SDS-PAGE and            SUB60 transformed by plasmid YEp96/UbWt, carrying
transferred to PVDF membrane following the method             the wild-type ubiquitin gene. The generation time of
described above. The membrane was probed with a               SUB60 cells lacking UBI4 and SUB60 transformants
mouse monoclonal antibody conjugated to peroxidase,           expressing the wild-type ubiquitin gene from plasmid was
namely anti-C-myc-Peroxidase (Roche), overnight at            2.5 and 2 h, respectively, which increased to 4 h in the
4 °C. The Western blot was developed with a chromo-           mutant UbEP42 in the absence of inducer CuSO4 and
genic substrate, 3,30 -diaminobenzidine, in the presence of   8 h and above after inducing cells with 25 and 50 lM
H2O2 and NiCl2 (Genei).                                       CuSO4 respectively (Fig. 2).

FEMS Yeast Res 14 (2014) 1080–1089                                             ª 2014 Federation of European Microbiological Societies.
                                                                                 Published by John Wiley & Sons Ltd. All rights reserved
1084                                                                                                                                                      A. Doshi et al.

                UbWt     UbEP42                                             KDa                             1           2                   3                  4
           10                                                      ***
Time (h)


           2                                                                Fig. 3. Western blot analysis of Cdc28 protein kinase. Lane 1,
           0                                                                protein molecular weight marker; lane 2, SUB60 cells; lane 3, SUB60
                       0 μM                  25 μM           50 μM
                                                                            cells transformed with Yep96/UbWt; lane 4, SUB60 cells transformed
                                                                            with Yep96/UbEP42.
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

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varied at from 0, to 25 to 50 lM to vary the expression levels of           offer the right background for checking the complemen-
UbWt and UbEP42. ***P < 0.001.                                              tation efficiency of mutant forms of ubiquitin under
                                                                            stress conditions. SUB60 cells transformed with YEp96/
                                                                            UbEP42 were exposed to heat stress and antibiotic stress
Table 2. Confocal microscopic studies of cell cycle progression with        to test if UbEP42 could complement successfully and
SUB62, SUB60, SUB60/UbWt and SUB60/UbEP42                                   rescue the cells under stress.
                                Percentage     Percentage   Percentage
Ubiquitin                       cells in       cells in     cells in        Heat stress complementation
variant                         G1 phase       S phase      G2 + M phases
SUB62                           68.2           21.3         10.5
                                                                            The complementation ability of UbEP42 under heat stress
SUB60                           65.0           22.1         12.9            was tested by subjecting SUB60 cells expressing UbEP42
SUB60/UbWt                      65.3           19.2         15.3            to heat stress at 40 °C for various intervals and returning
SUB60/UbEP42                    55.6           35.0          9.4            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
   Confocal microscopy was used to reassess our results                     severe effects than the untransformed SUB60 cells lacking
following the growth profile. The mid-log phase cultures                    the UBI4 gene, while control SUB60 cells transformed
of SUB62 cells, SUB60 cells and their transformants                         with the gene for UbWt were able to endure heat stress.
expressing UbWt and UbEP42 were observed using con-                         Thus, the result establishes the failure of UbEP42 in com-
focal microscopy (Forsburg & Nurse, 1991) and the                           plementing SUB60 cells under heat stress (Fig. 4).
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                       Antibiotic sensitivity test
S-phase leading to a reduction in growth rate. Hence, the                   Absence of the polyubiquitin gene UBI4 in SUB60 cells
results obtained by confocal microscopy confirm our                         makes them more sensitive to the antibiotic cycloheximide,
observations made earlier. As Cdc28 is known to regulate                    an effect nullified by expression of UbWt extrachromso-
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                                                 100
                                                                                                                              SUB 60      SUB 62        UbWt       UbEP42
cells expressing UbWt and UbEP42. From the results it is
                                                                            Percentage survival (%)

clear that UbEP42 expression has a negative influence on
Cdc28 level (Fig. 3).                                                                                  60


Complementation of stress-hypersensitive                                                               20

phenotype by UbEP42                                                                                     0
                                                                                                                0   4                8             12              16
The ubiquitin genes UBI1, UBI2 and UBI3 maintain basal                                                                      Incubation time (h)
levels of ubiquitin, which is required for normal function-
                                                                            Fig. 4. Effect of expression of UbEP42 on the survival of SUB60 cells
ing of cells. UBI4 supports the survival of cells under                     upon exposure to heat stress. SUB62, SUB60 and SUB60 transformed
stress conditions such as heat shock, starvation, UV dam-                   by YEp96/UbWt and YEp96/UbEP42. The cells were exposed to heat
age, amino acid analogs and antibiotics. Therefore,                         stress for different durations as shown in the graph and shifted to
SUB60 yeast cells which lack the UBI4 polyubiquitin gene                    30 °C.

ª 2014 Federation of European Microbiological Societies.                                                                            FEMS Yeast Res 14 (2014) 1080–1089
Published by John Wiley & Sons Ltd. All rights reserved
Studies on dosage dependent lethal ubiquitin mutant                                                                                                              1085

                    1      2         3   4    1                          2        3      4    Effect of the ubiquitin mutation on substrate
           SUB 62                                                                             protein turnover by the N-end rule and UFD
           SUB 60

   SUB 60/YEp96/
                                                                                              Varshavsky’s group designed ubiquitin fusions of b-galac-
           UbWt                                                                               tosidase to study protein degradation by the N-end
   SUB 60/YEp96/                                                                              rule (Johnson et al., 1992; Varshavsky, 1996) and UFD
                        –CYCLOHEXIMIDE            +CYCLOHEXIMIDE
                                                                                              pathways (Johnson et al., 1992, 1995). They observed
                                                                                              that b-galactosidase fusions of ubiquitin with stabilizing
Fig. 5. Effect of expression of UbEP42 on Saccharomyces cerevisiae                            N-terminal residues such as Met (M), as in Ub-Met-
(SUB60) under antibiotic stress. SUB60 cells transformed with YEp96/                          b-galactosidase, are cleaved by deubiquitinating enzyme
UbEP42 were grown on plates containing cycloheximide. SUB62 cells                             releasing a free Met-b-galactosidase which has a longer

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and SUB60 cells transformed with YEp96/UbWt were used as
                                                                                              half-life. b-Galactosidase fusions of ubiquitin with Pro at
controls. SUB60 and SUB60 transformed with UbEP42 fail to grow in
the presence of cycloheximide. Undiluted cultures (1), and threefold
                                                                                              their N terminus as in Ub-Pro-b-galactosidase are not
serial dilutions (2–4) were spotted on YPD plates containing 25 lM                            deubiquitinated. They therefore undergo polyubiquitina-
copper sulphate and 4 lg mL1 cycloheximide.                                                  tion 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 resi-
mally. To check if UbEP42 can complement in a similar                                         due of b-galactosidase. Changes in the level of b-galactosi-
fashion, the complementation study was extended further                                       dase activity can therefore reflect the effect of UbEP42 on
to cycloheximide stress. The two positive controls, SUB62                                     protein degradation. Thus, in the present study the two
and SUB60 transformed with the gene for UbWt, could                                           substrates Ub-Met-b-galactosidase and Ub-Pro-b-galacto-
resist the deleterious effect of cycloheximide. Expression of                                 sidase were chosen to understand the effect of UbEP42 on
the ubiquitin variant UbEP42 apparently renders SUB60                                         protein degradation. The results indicate that Met-b-galac-
cells more sensitive to cycloheximide as compared with un-                                    tosidase activity remained more or less unchanged with
transformed SUB60 cells lacking the UBI4 gene. The result                                     UbEP42 in the background (Fig. 6a). However, b-galacto-
suggests that one or more of the amino acid residue substi-                                   sidase activity increased with Pro-b-galactosidase in
tutions occurring in UbEP42 make it functionally impaired                                     SUB60 cells expressing UbEP42, as compared with those
through structural changes (Fig. 5).                                                          transformed by genes for UbWt and UbF45W. Conversely,

                                                                             65   (a)
                                                  Specific acitivity (103)




Fig. 6. Effect of UbEP42 on the degradation
                                                                                      SUB60   SUB62        60/M           62/M          60/M/Wt          60/M/42
of proteins in the background lacking UbI4.
SUB60, SUB62, and SUB60 transformed by                                       25
plasmids YEp96/UbWt and YEp96/UbEP42                                              (b)
expressing the two forms of ubiquitin, namely                                20                                                                              ***
                                                  Specific acitivity (103)

wild-type UbWt and UbEP42, were assayed for
b-galactosidase activity. These cells were also                              15
transformed by pUb23/Met expressing Ub-
Met-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                                      0
used as controls in the experiment were also
transformed by plasmid pUb23/Met and                                         –5
pUb23/Pro. ***P < 0.001.                                                              SUB60    SUB62        60/P           62/P          60/P/Wt           60/P/42

FEMS Yeast Res 14 (2014) 1080–1089                                                                             ª 2014 Federation of European Microbiological Societies.
                                                                                                                 Published by John Wiley & Sons Ltd. All rights reserved
1086                                                                                                                             A. Doshi et al.

                                                                                KDa     1      2       3       4       5     6



    1              2                  3               4     5
Fig. 7. Western blot analysis of b-galactosidase levels using anti-b-
galactosidase antibody. SUB60 cells transformed with pUb23/Met and               14.3
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

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                                                                        Fig. 8. Western blot showing ubiquitination profile. Lane 1, molecular
                                                                        weight marker. Position of bands and their molecular weights are
b-galactosidase activity of Ub-Pro-b-galactosidase in the               given for comparison. Lanes 2 and 4, uninduced SUB60 cells carrying
UbEP42 was is comparable to SUB60 expressing Ub-Pro-                    pUb221 and pUbEP42, respectively; lanes 3 and 5, SUB60 cells
b-galactosidase alone (Fig. 6b). This again indicated loss              carrying pUb221 and pUbEP42, respectively, in the presence of
                                                                        100 lM inducer; lane 6, untransformed SUB60 as negative control.
of function of UbEP42 in vivo. Hence, UbEP42 interferes
                                                                        Both UbWt and UbEP42 have N-terminal myc-tag and anti-myc
with the operation of the UFD pathway in S. cerevisiae.
                                                                        antibodies were used for Western blotting. Ubiquitin in SUB60 cells in
  Western blot analysis using anti-b-galactosidase anti-                lane 6 does not carry the tag and so no bands are seen, while a
bodies confirmed the results on protein stability obtained              characteristic polyubiquitination ladder is seen in lanes 3 and 5.
with b-galactosidase assays (Fig. 7).                                   UbEP42 can therefore be incorporated into polyubiquitin chains.

Incorporation of UbEP42 into polyubiquitin
                                                                        mutant has a negative effect on the degradation of
                                                                        Ub-Pro-b-galactosidase, implying that the polyubiquitin
High levels of Pro-b-galactosidase activity in SUB60 with               chain of UbEP42 is not recognized by proteasomes for
UbEP42 expression indicated two possibilities: either                   degradation. The protein also could not rescue the
UbEP42 is not recognized by the ubiquitination system or                cells from heat stress, probably due to its thermal
it is recognized by components of the ubiquitination sys-               instability.
tem 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 con-
jugated to candidate proteins and extended into poly-                   Ubiquitin serves as an example of a protein that strikes
ubiquitin chains, in a processing step required for the                 an optimal balance between structure and function early
specific degradation of candidate proteins (Ellison &                   on evolution, undergoing just three changes in sequence
Hochstrasser, 1991). In the present experiment, UbEP42                  from yeast to human (Mishra et al., 2009). Ubiquitin
was similarly tagged with c-myc to follow its incorporation             interacts with innumerable enzymes in the process of
into polyubiquitin chains in SUB60. c-myc-tagged UbWt                   ubiquitination and deubiquitination of a variety of candi-
was used as a positive control.                                         date proteins belonging to diverse pathways. With the
   Immunoblot analysis of c-myc-tagged UbEP42 indi-                     benefit of hindsight, it appears that ubiquitin’s chances of
cated that UbEP42 is incorporated into polyubiquitin                    accumulating mutations were obliterated to avoid coevo-
chains, suggesting that the conjugation is not affected by              lution of interactive partners to maintain their interac-
mutations (Fig. 8). Hence, the function that could be                   tions with an evolving ubiquitin. However, this feat of
affected is the selective and regulated degradation of pro-             conservation did not leave any room for understanding
teins. This result also explains the lethal effect of UbEP42            the functional role of individual residues in ubiquitin.
overexpression. The overexpression of UbEP42 far exceeds                   Sloper-Mould et al. (2001) observed that there are
the basal levels of wild-type ubiquitin in SUB60. Proteins              three functionally important surfaces in ubiquitin. They
ubiquitinated with UbEP42 probably accumulate in the                    also established that Ser20Ala and Ala46Gly substitutions
system, causing lethality.                                              do not have any significant influence over growth, tem-
   Together our results show that UbEP42 can be used                    perature and cold sensitivity of S. cerevisiae. Besides,
normally by the cell for polyubiquitination. However,                   studies on ubiquitin binding domains (UBDs) pointed
UbEP42 failed to complement SUB60 cells under heat                      to two types of interactions between ubiquitin and
stress and antibiotic stress. It was observed that the                  UBDs, which involved other residues (Harper & Schul-

ª 2014 Federation of European Microbiological Societies.                                                    FEMS Yeast Res 14 (2014) 1080–1089
Published by John Wiley & Sons Ltd. All rights reserved
Studies on dosage dependent lethal ubiquitin mutant                                                                              1087

man, 2006; Dikic et al., 2009; Komander, 2009; Bomar          side chain of Ile61 is also buried in a hydrophobic
et al., 2010).                                                pocket formed by Ala46 and Leu67 of the wild-type
   Previously, our laboratory has focused on the parallel     protein and hence the replacement hydrophobic residues
b-bulge of ubiquitin to gain insight into its structure–      by polar residues in Ala46Ser and Ile41Thr are meant to
function relationships (Mishra et al., 2009, 2011; Sharma     complement each other. The structure of ubiquitin is
& Prabha, 2011; Prabha et al., 2012). The residues in the     stabilized by its globular shape with a hydrophobic core
b-bulge were substituted using site-directed mutagenesis.     and extensive hydrogen bonding. These mutations suc-
The amino acid residue replacements were chosen to pre-       cessfully explain its functional impairments and the
serve the structure of ubiquitin intact and the functional    observed decrease in thermal stability of UbEP42. The
integrity of the resultant variants was studied. Although     mutations described here, Ser20Phe and Ala46Ser, are
this approach is highly specific, it is limited only to the   different from those studied by Sloper-Mould et al.

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site of choice and the residue chosen for replacement. On     (2001) and the residues Leu50 and Ile61 did not form
the other hand, generation of mutants using in vitro evo-     part of the set of mutations chosen by them.
lution gives rise to a library of mutations. Selection and       Although we wanted to study the role of individual res-
characterization of individual mutations generated using      idues in ubiquitin using single mutants, the UbEP42
error-prone PCR in such cases can expand our knowledge        mutant generated by error-prone PCR happened to carry
more rapidly.                                                 four amino acid substitutions (Prabha et al., 2010). Char-
   In the present study, we have characterized the dos-       acterization of individual mutations of UbEP42 and their
age-dependent ubiquitin lethal mutant UbEP42. The             combinations may reveal two important aspects of these
mutant of ubiquitin UbEP42 has four mutations, which          mutations. First, they highlight the functional contribu-
render the protein thermally unstable. The hydropathy         tions made by these four residues to ubiquitin biology.
indices and secondary structural preferences of the origi-    Secondly, they unravel any cumulative or compensatory
nal and substituting residues are widely different in all     influences the mutations are exerting over each other in
four cases (Table 1). Low level expression of UbEP42 in       UbEP42. Third and most importantly, expressing UbEP42
SUB60 cells with UBI4 deletion did not help them with-        in higher organisms under tissue-specific promoters may
stand exposure to cycloheximide. The presence of              lead to targeted removal of proteins where derailed pro-
UbEP42 in the cellular ubiquitin pool impedes the deg-        tein degradation is the underlying cause for pathogenesis.
radation of the substrates of the UFD pathway. Incorpo-          In conclusion, a dosage-dependent lethal mutation of
ration of UbEP42 into polyubiquitin chains rules out          ubiquitin UbEP42, despite being incorporated into poly-
deficiency of ubiquitin as a likely cause for lethality,      ubiquitin chains, does not compensate for the deficiency
unlike the case with strain SUB60. Incorporation of a         of UBI4 in S. cerevisiae strain SUB60, leaving it suscepti-
functionally inept ubiquitin leading to failure of degrada-   ble to heat stress and antibiotic stress. Under normal con-
tion of proteins is a likely reason for the lethality         ditions overexpression of UbEP42 in UBI4-deleted strains
observed in this case.                                        caused cell lysis, while low levels led to a competitive
   In UbEP42 the substitutions of Ser20 to Phe and            inhibitor-like effect that slowed growth of the organism
Ala46 to Ser occurring in type I and type III turns,          considerably. Furthermore, UbEP42 has reduced the level
respectively (Vijay-kumar et al., 1987), have similar pro-    of Cdc28 protein kinase. Besides, the UbEP42 background
pensities for the secondary structures. Furthermore,          hampers the degradation of chimeric fusions of ubiquitin
despite large differences in their hydropathy indices,        degraded by the UFD pathway. Harnessing these observa-
these substitutions could be accommodated due to their        tions made with UbEP42 may assist the development of
location on the surface of the protein, as suggested from     novel approaches in the treatment malignancies and neu-
our more recent observations (M. Sharma, A. Doshi and         rodegenerative disorders.
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      C.R.P. is grateful to the University Grants Commission
have a considerable negative impact on the structure          for the Major Research Project [No. F.33-225/2007 (SR)].
and stability of the protein as Pro has very low propen-      C.R.P. thanks Professor Mark Searle and Professor Daniel
sity for the b-strand. Furthermore, both Ala46Ser and         Finley for providing the plasmids and strains necessary
Leu50Pro occur closer to the two functionally important       for the study. We thank the DBT-ILSPARE facility of The
residues Ile44 and Lys48. The substitution of Ile61 by        M. S. University of Baroda, India, for use of the confocal
Thr affects one of the residues protected early during        microscopy facilities. The authors have no conflict of
the refolding of ubiquitin (Briggs & Roder, 1992). The        interest to declare.

FEMS Yeast Res 14 (2014) 1080–1089                                             ª 2014 Federation of European Microbiological Societies.
                                                                                 Published by John Wiley & Sons Ltd. All rights reserved
1088                                                                                                                   A. Doshi et al.

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FEMS Yeast Res 14 (2014) 1080–1089                                                    ª 2014 Federation of European Microbiological Societies.
                                                                                        Published by John Wiley & Sons Ltd. All rights reserved
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