Encore facilitates SCF-Ubiquitin-proteasome-dependent proteolysis during Drosophila oogenesis
Johanna Talavera Ohlmeyer and
Trudi Schüpbach*
HHMI/Molecular Biology Department, Princeton University, Princeton, NJ
08544, USA
*
Author for correspondence (e-mail:
gschupbach{at}molbio.princeton.edu)
Accepted 10 September 2003
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SUMMARY
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Exit from the cell cycle requires the downregulation of Cyclin/Cdk
activity. In the ovary of Drosophila, Encore activity is necessary in
the germline to exit the division program after four mitotic divisions. We
find that in encore mutant germaria, Cyclin A persists longer than in
wild type. In addition, Cyclin E expression is not downregulated after the
fourth mitosis and accumulates in a polyubiquitinated form. Mutations in genes
coding for components of the SCF pathway such as cul1, UbcD2 and
effete enhance the extra division phenotype of encore. We
show that Encore physically interacts with the proteasome, Cul1 and Cyclin E.
The association of Cul1, phosphorylated Cyclin E and the proteasome 19S-RP
subunit S1 with the fusome is affected in encore mutant germaria. We
propose that in encore mutant germaria the proteolysis machinery is
less efficient and, in addition, reduced association of Cul1 and S1 with the
fusome may compromise Cyclin E destruction and consequently promote an extra
round of mitosis.
Key words: Mitosis, Encore, Oogenesis, Cyclin E, SCF, Proteolysis, Drosophila
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Introduction
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In Drosophila, the development of the egg starts at the anterior
tip of the ovary, in the germarium, where the germline stem cells divide to
produce a cystoblast and a self-renewing stem cell. Each cystoblast undergoes
four mitotic divisions with incomplete cytokinesis. The resulting 16 cells of
each egg chamber are connected by intercellular bridges called ring canals
(Fig. 1A). One of the cells
with four ring canals develops into the oocyte and the rest give rise to nurse
cells. Each of the four mitoses is oriented and synchronized by the fusome; a
germline specific organelle composed of membrane and cytoskeletal proteins
(Storto and King, 1989
;
Lin et al., 1994
;
Deng and Lin, 1997
). After
each division, the fusome grows by fusion of ER-Golgi type vesicles and
extends through the ring canals in order to connect all the cells of the cysts
(de Cuevas and Spradling, 1998
;
Leon and McKearin, 1999
)
(Fig. 1A). The mechanism by
which the number of cyst mitoses is limited to four has not been fully
elucidated. However, the studies of various mutations suggest that the fusome
plays a role in regulating the timing, the synchronization, and perhaps the
exit from the cell cycle in the germarium. Several genes have been implicated
in the regulation of germline division. Mutations in genes coding for
components of the fusome such as hu-li tai shao (hts),
and ß spectrin (Yue
and Spradling, 1992
; Deng and
Lin, 1997
; Lin et al.,
1994
; de Cuevas et al.,
1996
), and Dynein heavy chain (Dhc64)
(McGrail and Hays, 1997
)
result in egg chambers that contain less than 16 germline cells and often lack
an oocyte. The integrity of the fusome is compromised and the resulting number
of cells in these mutant egg chambers is variable and not always a factor of
2n as in the wild-type cyst. Mutations in genes encoding proteins
that associate with the fusome such as bag of marbles (bam)
or genes required for proper association of Bam to the fusome such as
benign gonial cell neoplasm (bcgn) result in mutant egg
chambers that are tumorous and filled with proliferating cells
(Lavoie et al., 1999
;
McKearin and Ohlstein, 1995
).
Mutations in the ovarian tumor (otu) gene that produce
tumorous egg chambers have fragmented fusomes
(King and Storto, 1988
).
Overexpression or loss-of-function mutations in a third group of genes such as
Cyclin A, Cyclin B, Cyclin E and mutations in the gene encoding the
E2 Ubiquitin conjugating enzyme UbcD1 lead to the production of cysts with 32
or 8 cells (Lilly et al.,
2000
). These genes do not affect fusome integrity and thus timing
and spatial characteristics of cell division appear to be intact. The
encore gene belongs to this group of genes, its product is necessary
for exit from mitosis. Loss of Encore activity results in egg chambers
containing 32 rather than 16 cells
(Hawkins et al., 1996
;
Van Buskirk et al., 2000
).
Mutations in the encore gene produce additional phenotypes, which
show differential temperature sensitivity. encore mutant females
raised at 18°C produce egg chambers with 16 cells, but they give rise to
ventralized eggs (Hawkins et al.,
1997
). The extra cell division phenotype is only observed when
encore mutant females are raised at high temperatures (25-29°C).
The encore gene encodes a 200 kDa protein with no homolog of a
defined biochemical function (Van Buskirk
et al., 2000
). In this work we analyze the mechanism by which
Encore promotes exit from the cell cycle after four germline mitoses.

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Fig. 1. Cyclin A protein expression in the germarium. (A, left) The four mitotic
divisions of the cystoblast and its stereotypic relationship with each other
and with the fusome (red). (A, right) The different regions of the
Drosophila germarium. The germline stem cells, and dividing
cystoblast comprise region 1. Region 2A contains post-mitotic 16 cell-cysts
(green). The boundary between regions 2A and 2B is marked by the follicle
cells migrating inwards to envelop the cyst and to form the egg chamber. The
red lines represent the spectrosome in the germline stem cells and the fusome
connecting all the cells in the dividing cystoblast and postmitotic cyst.
Region 3 of the germarium is characterized by the budding of newly formed egg
chambers. (B-G) Expression of Cyclin A (red) and the mitotic marker
phosphohistone 3 (green) in wild-type and in encore mutant germaria.
(B-D) In wild-type ovaries, Cyclin A is expressed in the stem cells and
dividing cystoblast in region 1 of the germarium. It rapidly declines in
post-mitotic cysts. (E-G) In encore mutant germaria, Cyclin A
expression persists after the end of mitosis. The arrow shows a cyst positive
for Cyclin A in a posterior area of the germarium.
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Cell cycle progression is controlled by a series of cell cycle dependent
kinases (Cdk). Cdk activity is carefully regulated by the levels of the Cyclin
subunits, by Cdk inhibitors (CKI) and by post-translational modification of
the Cdk subunit through both activating and inactivating phosphorylation
(Desai et al., 1995
;
Nakayama et al., 2001
).
Transition from G1 to S phase depends on Cdk2/Cyclin E activity, and on the
timely destruction of the Cdk2/Cyclin E inhibitor p27. The Drosophila
p27 homologue, Dacapo, is required for exit from the cell cycle in the embryo
and eye imaginal disc (Lane et al.,
1996
; de Nooij et al.,
1996
). In addition, exit from the cell cycle requires destruction
of the cyclins by the ubiquitin-proteasome system (UPS)
(Ciechanover et al., 2000
;
Varshavsky et al., 2000
). The
addition of ubiquitin requires three different activities; the ubiquitin
activating enzyme (E1), the ubiquitin conjugating enzyme (E2) and the
ubiquitin ligase enzyme (E3). The ubiquitinated protein bound to E3 is
presented to the proteasome, isopeptidase activities in the 19S-recognition
particle (RP) of the proteasome cleave the ubiquitin tail, the protein is
unfolded and finally destroyed by the proteasome 20S-core particle (CP)
(Verma et al., 2002
;
Cope et al., 2002
). There are
two E3 enzyme complexes that regulate the cell cycle progression
(Tyers and Jorgensen, 2000
;
Koepp et al., 1999
). The
APC/cyclosome regulates progression from G2 to M phase transition. The SCF
complex regulates the G1 to S phase transition. The SCF complex is composed of
Skp/Cullin/Rbx1 and F-box proteins and controls substrate ubiquitination via
an interaction between the F-box component and the phosphorylated target
protein. In Drosophila and mammalian systems, mutations in the
Cul3 and Ago genes cause the accumulation of Cyclin E, entry
to S-phase and doubling of cell number
(Moberg et al., 2001
;
Singer et al., 1999
). Thus,
proper regulation of the destruction machinery is important for maintaining
normal levels of Cyclin E and assuring proper cell cycle progression. The work
presented in this paper demonstrates that the encore gene product
associates with the SCF-UPS and is required for proper exit from germline
mitosis. The failure to downregulate Cyclin E after four cell divisions in
conjunction with an accumulation of Cyclin A protein provide the conditions to
promote an extra cell division. We show that Encore can bind to Cul1, Cyclin
E-Ub(n) and the proteasome. We also demonstrate that Cul1 and the
proteasome 19S-RP subunit S1 are associated with the fusome and these
associations are very much attenuated in encore mutant ovaries. We
propose that as a direct consequence, Cyclin E is not degraded properly, its
activity is misregulated and the cyst undergoes one extra cell division.
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Materials and methods
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Stocks and reagents
The encore alleles used in this study were the EMS induced alleles
encQ4 and encRI and the P-element
allele encBB (Hawkins
et al., 1996
). The cyclin A alleles, l(3)183
(Lehner and O'Farrell, 1989
)
and l(3)C8LRI (Lehner et al.,
1991
), and stocks containing the HS-cylin A or
HS-cyclin E transgenes were provided by C. Lehner
(Knoblich and Lehner, 1993
;
Knoblich et al., 1994
). The
stock containing the HS-rux transgene was obtained form L. Zipursky
(Thomas et al., 1997
). H.
Richardson and the Developmental Studies Hybridoma supplied the polyclonal
Cyclin A antibody. Monoclonal and polyclonal antibodies against
Drosophila Cyclin E were obtained from C. Lehner, H. Richardson, M.
A. Lilly and purchased from Santa Cruz Biotechnology. V. Fillipov provided the
polyclonal antibody against Drosophila Cul1. The S1 and LMP7
antibodies were purchased from Upstate Biotechnology. The phosphorylated
Cyclin E antibody was purchased from Santa Cruz Biotechnologies. The
cyclin E alleles (05206 and k05007), and
UbcD2 (k13206), effete (S1782 and
8) and cul1 [Df(2R)CA53 and lin19] alleles
were supplied by the Bloomington Stock Center.
Antibody staining
Ovaries of well-fed flies were dissected in ice-cold PBS and fixed with 4%
paraformaldehyde in PBST for 10 minutes. Fixed ovaries were washed in PBST,
blocked with 5% BSA in PBST and incubated in the primary antibody at 4°C
overnight. The fluorescent secondary HRP antibodies (Vector) were used at 1 to
1000 dilution. The Cul1 and S1 antibody staining was performed with the
following modifications. After dissection, the ovaries were incubated in
heptane-PBST0.3% Tween) for 10 minutes, washed for 1 hour in PBST at 4°C,
blocked for 2 hours at 4°C, followed by incubation with the primary
antibody to 1:1000 dilution overnight at 4°C. The ovaries were then washed
three times with PBST and fixed as usual at room temperature. Images of
stained samples were collected with a Zeiss Confocal Microscope.
Western blots and immunoprecipitation assays
Ovary dissection was performed in ice-cold 50 mM Tris buffer containing
protease inhibitors (Roche), 250 µM NEM and 10 mM MG132 and as much of the
vitellarium as possible was carefully dissected away. The germarium-enriched
region was then transferred to ice cold Tris buffer, homogenized, centrifuged
and sample buffer was added. Super Signal Chemiluminescent kit (Pierce) was
used for signal detection. For immunoprecipitation assays, the
germaria-enriched extracts were precleared for 30 minutes with AG beads
(Pharmacia), followed by a 1 hour incubation at room temperature with the
precipitating antibody and AG beads. The beads were then washed six times and
sample buffer added and run in a 7%Tris-acetate Nupage polyacrylamide gel
(Invitrogen).
Proteasome activity assays
Germaria-enriched extracts were prepared in ice-cold 50 mM Tris buffer. To
start the reaction, 2 mM DTT, 5 mM MgCl2, 2 mM ATP and 10 µM of
the fluorescent peptide succinyl-leu-leu-val-tyrosine-7-amido-methylcoumarin
(Succ-LLVY-MCA) was added. The reaction was incubated at 29°C and aliquots
were collected every 20 minutes and added to 200 µl of 1%SDS to stop the
hydrolysis reaction. Peptide hydrolysis was monitored measuring by fluorescent
emission at 440 nm.
Ubiquitination reaction
Germaria-enriched extracts were prepared in Tris buffer containing protease
inhibitors. To start the ubiquitination reaction 10 mM MG132, 0.1 µg/µl
Ubiquitin aldehyde, 5 µg/µl Ubiquitin, 5 mM ATP
S, 20 mM
MgCl2, 2 mM DTT and 2 µg of histidine-tagged p27 was added,
followed by 2-8 hours incubation at 37°C. Polyubiquitinated p27 was
affinity purified using nickel-agarose beads. The de-Ubiquitination reaction
was performed by adding 20 mM MgCl2, 2 mM DTT, 10 mM Creatine
phosphate and 8 µg Creatine Kinase and 5 mM ATP to germaria-enriched ovary
extract. The reaction was incubated at 37°C and aliquots were taken every
20 minutes. The addition of sample buffer stopped the reaction.
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Results
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Mutations in the encore gene cause the accumulation of
Cyclin A protein in the Drosophila germarium
In wild-type ovaries, Cyclin A protein is expressed in a cell
cycle-dependent manner (Lilly et al.,
2000
). The Cyclin A protein is detected in the stem cells, and in
dividing cystoblasts. Its expression declines rapidly in post-mitotic cysts
(Fig. 1B-D). In encore
mutant females raised at 29°C Cyclin A protein expression lingers longer
than in wild-type germaria (Fig.
1E-G) and is detected in cysts located in a posterior area not
associated with Phospho-histone3 expression. This suggests that Cyclin A
protein remains present after mitoses have stopped. The persistence of Cyclin
A expression in the germarium may indicate that proper Cyclin A protein
turnover is defective. In the Drosophila embryonic cellular
blastoderm Cyclin A distribution is very dynamic and accumulates in the
cytoplasm during interphase, in the nucleus during prophase and is degraded
during metaphase (Lehner and O'Farrell,
1989
; Whitfield et al.,
1990
). Immunohistochemical assays also show a dynamic Cyclin A
subcellular localization in the germarium, which seems to depend on the cell
cycle stage. Cyclin A levels in the cytoplasm increase to fill the cyst
completely. In these cysts, Cyclin A is also observed in transient association
with the fusome during late prophase/metaphase of the cell cycle
(Lilly et al., 2000
)
(Fig. 2A-C). In dividing cells,
Cyclin A is degraded at metaphase and there is no detectable Cyclin A in
anaphase and telophase. Cytoplasmic accumulation and nuclear localization of
Cyclin A in encore mutant germaria is comparable with wild type. The
transient association of Cyclin A with the fusome, however, is prolonged as we
can observe Cyclin A in the fusome in a posterior position of the germarium
(Fig. 2D-F, arrow). The
accumulation of Cyclin A protein in encore mutant germaria can also be
observed in western blots (Fig.
2H). Increased levels of Cyclin A protein are observed in
germaria-enriched extracts from encore mutant ovaries compared with
wild type. These data suggest that encore mutations at the
restrictive temperature of 29°C promote the accumulation and/or prevent
the timely turnover of Cyclin A protein in the germarium.

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Fig. 2. Cyclin A promotes the encore extra division phenotype. (A-C) In
wild-type germaria Cyclin A (red) associates with the fusome (green stained
with phalloidin for actin) in dividing cystoblasts (arrow and inset). (D-F)
Association of Cyclin A with the fusome in encore mutant germaria
persists in posterior regions of the germarium (arrow and inset). (G)
encore mutant females raised at 25°C produce 55% of the egg
chambers with 32 cells. Reducing Cyclin A gene dose has no effect on
the encore cell division phenotype. However overexpression of the
HS-rux transgene in an encore mutant background reduces the
32 cells egg chamber phenotype from 55 to 25%. Additional reduction of
Cyclin A gene dose results in 20% of the egg chambers containing 32
cells. (H) Western blot showing that the amount of Cyclin A protein in
encore mutant extract is increased compared with the wild-type
extract. Equivalent amounts of germaria-enriched extract were loaded in both
lines as seen with the protein dye ponceau (pink).
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Reduction of Cyclin A protein by overexpression of the Cyclin A
inhibitor Roughex suppresses the encore extra division phenotype
Overexpression of a Cyclin A transgene under the control of heat
shock promoter results in an extra mitotic division in only 3% of the egg
chambers. However, expression of a stable form of Cyclin A increases the
number of egg chambers containing 32 rather than 16 cells to 17%
(Lilly et al., 2000
). In our
hands, the expression of the HS-Cyclin A transgene causes an extra
mitotic division in 4% of egg chambers. Given these results and the observed
accumulation of Cyclin A protein in encore mutant germaria, we wanted
to find out whether Cyclin A is responsible for the extra mitotic division
phenotype. Reduction of Cyclin A gene dose by half in an
encore mutant background does not suppress the extra division
phenotype (Fig. 2G). One reason
for this result could be that one copy of the Cyclin A gene might
produce enough protein to allow an extra cell division. Another possibility is
that reduction of Cyclin A gene dose is compensated by turning on
feedback mechanisms that affect the production or stability of Cyclin A. In
order to circumvent this problem we took a different approach to decreasing
the amounts of Cyclin A protein by over-expressing the Cyclin A inhibitor,
Roughex (Rux). The rux gene product binds to Cyclin A and this
complex is then transported to the nuclei where it is destroyed via the UPS
(Sprenger et al., 1997
;
Thomas et al., 1997
;
Foley et al., 1999
;
Avedisov et al., 2000
).
Overexpression of Rux using a HS-rux transgene gives rise to a
reduced number of mitotic divisions. Similarly, in Drosophila
embryonic ectoderm and imaginal discs
(Thomas et al., 1997
),
expression of the HS-rux transgene at 37°C reduces the number of
mitoses. The expression of the HS-rux transgene alone or in an
encore heterozygous mutant background at 25°C had no effect on
mitosis. Flies expressing the HS-rux transgene in an encore
mutant background were raised at 25°C. In this experiment about 55% of the
encore mutant control ovaries contained 32 cell egg chambers
(Fig. 2G). The mild expression
of Rux resulted in the suppression of the extra mitotic division phenotype
such that only 25% of the egg chambers showed the encore phenotype.
The extent of the HS-rux suppression did not vary significantly when
in addition to expressing HS-rux, cyclin A gene dose was reduced by
half. Thus, it seems that the extra Cyclin A protein present in the
encore mutant germarium contributes to the promotion of an extra
mitotic division.
Cyclin E protein expression is altered in encore mutant
germaria
In hypomorphic mutations of the Cyclin E gene, 30% of the egg
chambers have only eight cells (Lilly and
Spradling, 1996
). Conversely, expression of a heat inducible
cyclin E transgene induces entry to S phase and results in an extra
round of mitosis in the Drosophila embryo and eye imaginal disc
(Knoblich et al., 1994
;
Richardson et al., 1995
). When
we express the HS-cyclin E transgene, a modest 6% of egg chambers
produce an extra cell division. To assess Cyclin E protein expression in
encore mutant ovaries, immunolocalization experiments were carried
out on flies raised at 29°C. In wild-type germaria, Cyclin E protein is
expressed in the nuclei of the germline stem cells, cystoblasts and dividing
cysts (Fig. 3A). The protein
levels are dramatically reduced after mitosis ends. There is no Cyclin E
expression in postmitotic cysts of region 2A and it is absent in region 2B of
the germarium. Cyclin E protein expression resumes in region 3 and persists
throughout the rest of oogenesis. However, this second phase of Cyclin E
protein expression is no longer synchronized as not all the cells in the egg
chamber express Cyclin E simultaneously
(Fig. 3A). This pattern of
expression is in accordance with BrdU incorporation experiments
(McKearin and Ohlstein, 1995
)
that indicate the requirement for Cyclin E during S phase of the mitotic cycle
and of the endocycle. In encore mutant ovaries, Cyclin E protein is
expressed throughout the germarium, indicating that the mechanism of
downregulation of Cyclin E at stage 2A and 2B is defective
(Fig. 3B). Cyclin E is degraded
during S phase in the cell cycle (Follette
and O'Farrell, 1997
). Thus, in wild-type germaria, its expression
oscillates and not all the cysts in region 1 express Cyclin E simultaneously
(Fig. 3A, inset). In
encore mutant germaria, all cysts express some Cyclin E, indicating
that at each cell division Cyclin E degradation is affected
(Fig. 3B, inset). The
unsynchronized expression of Cyclin E in region 3 and later is comparable with
wild-type ovaries. The Cyclin E protein expression in wild-type germaria
suggests that cessation of mitosis in the ovary requires Cyclin E
downregulation. Thus, we tested if the persistent expression of Cyclin E in
encore mutant germaria can promote the extra division phenotype.
Double mutant females homozygous for encore and heterozygous for
Cyclin E were raised at 29°C. We observed that indeed the
encore extra division phenotype is suppressed from 70% to 10% in the
double mutant females ovaries (Fig.
3C). The suppression of the encore phenotype is more
pronounced when Cyclin E gene dose is reduced than when Cyclin A
activity is reduced. It has been shown that Cyclin E overexpression can
promote the accumulation of Cyclins A and B in the Drosophila
embryonic ectoderm without affecting RNA levels
(Knoblich et al., 1994
;
Lane et al., 1996
). As S-phase
progresses and Cyclin E expression increases, the CycE/Cdk2 complex promotes
the destruction of the Rux protein (Thomas
et al., 1997
; Sprenger et al.,
1997
; Avedisov et al.,
2000
) and allows Cyclin A to accumulate in the cell. We tested
whether the accumulation of Cyclin A in encore mutant germaria is a
consequence of the abnormal expression of Cyclin E. Reduction of cyclin
E gene dose by half in an encore mutant background clearly
reduces the accumulation of Cyclin A protein
(Fig. 3D). Given these results,
we propose that the persistent expression of Cyclin E in the encore
mutant germaria causes the accumulation of Cyclin A. Reducing Cyclin E dose
brings Cyclin A expression down to more normal levels resulting in suppression
of the extra mitotic division. Because reducing Cyclin A activity levels has
only a partial effect in suppressing the extra division, we believe that the
extra round of mitosis produced by Encore is promoted by the joint effects of
accumulating Cyclin E and Cyclin A proteins.

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Fig. 3. Expression of Cyclin E in the germarium. (A,B) Spectrin (red) marks the
spectrosome in the germline stem cell and the fusome in the developing cyst.
(A) Cyclin E protein expression (green) in the wild-type ovary is observed in
region 1 of the germarium and sharply decreases in regions 2A and 2B.
Asynchronous expression of Cyclin E resumes in region 3 and persists in the
vitellarium. In wild-type germaria, Cyclin E expression oscillates strongly
(inset): some cysts show Cyclin E expression (arrow), while other cysts are
depleted of Cyclin E protein (arrowhead). (B) In encore mutant
germaria, Cyclin E expression is observed throughout the germarium. There is
no downregulation in region 2A/B. In encore mutant germaria, all
cysts express some Cyclin E protein all the time (inset). (C) Reducing
cyclin E gene dose in an encore mutant background suppresses
the encore 32-cell egg chamber phenotype from 70 to 10% in females
raised at 29°C. (D-F) Western blots of germaria-enriched extracts. (D) The
accumulation of Cyclin A observed in encore mutant females can be
partly restored to more normal levels by reducing cyclin E gene dose.
The blot was stained with the protein dye ponceau (pink) as loading control.
(E) The inhibition of proteolysis (Pase Inh.) or/and 19S-RP isopeptidase
activity (NEM/Llnl) reveals that in encore mutant extracts Cyclin E
accumulates as a high molecular form protein (arrowheads). The blot was
stained with the protein dye ponceau (pink) as loading control. (F)
Immunoprecipitation assays using anti-Drosophila Cyclin E antibodies
followed by IB using anti-Ubiquitin antibodies shows more Cyclin
E-Ubn in the encore mutant extract lane compared with wild
type. The reciprocal immunoprecipitation assay shows more Cyclin
E-Ubn in the encore mutant lane. Extract concentration of
the starting material is shown stained with Coloidal blue Coomassie. The
control lane (co) represents immunoprecipitation using wild-type extracts and
an unrelated antibody raised in the same animal as the immunoprecipitation
test.
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Cyclin E protein turnover is defective in the encore mutant
germarium
An important feature of the cell cycle is the tight regulation of the
Cyclin/Cdk complexes by the rapid and timely destruction of the cyclin
partner. We wanted to test whether the persistence of Cyclin E protein in
encore mutant germaria results from improper degradation. Female
flies were raised at 29°C and western blots of extracts enriched for
germaria and previtellogenic egg chambers were performed. These experiments
revealed some differences between the expression of Cyclin E in the wild-type
and encore mutant ovaries (Fig.
3E). In Drosophila, the Cyclin E transcript
encodes two proteins: the zygotic and the maternal forms of Cyclin E, which
are products of differential splicing
(Richardson et al., 1993
;
Jones et al., 2000
). The
predicted molecular weight for the maternal Cyclin E protein is 78 kDa and for
the zygotic Cyclin E is 60 kDa. Western blots using polyclonal antibodies
against Cyclin E show that extracts from encore mutant ovaries
accumulate high molecular weight forms of Cyclin E
(Fig. 3E, arrowheads). Extracts
made in the absence of protease inhibitors show little protein and no
difference in Cyclin E expression between the encore mutant and
wild-type extracts (arrows). The addition of protease inhibitors results in a
stronger signal in both wild-type and encore and reveals a clear
difference in Cyclin E levels between encore mutant and wild-type
extracts. Cyclin E accumulates as high molecular weight protein in the
encore mutant lane compared with wild-type extract lane. This
observation suggests a slower rate of Cyclin E degradation in the
encore mutant extract. The addition of isopeptidase inhibitors
further protects these high molecular weight forms of Cyclin E. NEM and Llnl
inhibit the action of the proteasome 19S-RP isopeptidases that de-ubiquitinate
substrates. In order to confirm that the high molecular weight bands are
Cyclin EUbn, germaria-enriched extracts were prepared in the
presence of protease inhibitors, NEM and Llnl. Immunoprecipitation assays were
performed using antibodies against Cyclin E followed by immunoblot (IB) with
antibodies against Ubiquitin (Fig.
3F). Cyclin E in the wild-type extract and the accumulated Cyclin
E in the encore mutant extract are recognized by antibodies against
ubiquitin. The reciprocal immunoprecipitation using antibodies against
Ubiquitin followed by IB with antibodies against Cyclin E confirm that the
rate of degradation of Cyclin E-Ubn in the encore mutant
extract is compromised.
Mutations in genes encoding SCF pathway components enhance
encore's mitotic phenotype
Our results suggest that proper destruction of Cyclin E requires Encore
activity. In order to test this idea further, we generated double mutant flies
using encore mutations and mutations in genes that encode for
components of the SCF ubiquitination pathway. Mutations in the cul1
and archipelago (Ago) genes result in the accumulation of
Cyclin E in mammals and in the Drosophila eye
(Wang et al., 1999
;
Dealy et al., 1999
;
Moberg et al., 2001
;
Koepp et al., 2001
). As
expected, reduction of the cul1 gene dose in an encore
mutant background enhances the extra division phenotype from 27 to 65% at the
mildly restrictive temperature of 25°C
(Fig. 4G). Moreover
cul1 mutations enhance the encore phenotype at room
temperature from 3% to 44%. Thus, the reduction of Encore activity sensitizes
the system such that even at room temperature, the proteolysis machinery can
no longer ensure the destruction of Cyclin E when cul1 gene dose is
reduced. Similar results were obtained with mutations in the Ubiquitin ligase
component UbcD2 and effete (UbcD1)
(Fig. 4G). Reducing
cul1 gene dose in an encore heterozygous background produces
only 16-cell egg chambers. We propose that the reduction of Cul1 results in
decreased degradation efficiency and accumulation of Cyclin E. In this
situation, Encore is required to facilitate the destruction of the surplus
Cyclin E. As expected for a component of the SCF-UPS, the Drosophila
Cul1 protein is a nuclear protein and is expressed throughout oogenesis
(Filippov et al., 2000
). Cul1
protein expression in the germaria of wild-type females raised at room
temperature or 29°C, shows strong localization to the fusome
(Fig. 4A-C, inset). Unlike
Cyclin A, Cul1 association with the fusome is not transient. Cul1 protein is
observed in the germline stem cells in association with the spectrosome
(Fig. 4A-C, arrow). During
cystoblast division and up to region three of the germarium Cul1 can be seen
in association with fusome. In encore mutant germaria of flies raised
at 29°C, the association of Cul1 with the fusome is disrupted. There is
more nuclear Cul1 staining in encore mutant germaria compared with
the wild type (Fig. 4D-F,
inset). We have performed western blots using germarium-enriched extract and
do not observe any difference between the levels of Cul1 in wild-type and
encore mutant extracts (data not shown). This suggests that Encore
activity is required for proper Cul1 localization to the fusome and that Cul1
association with the fusome may be important for proper Cyclin E processing.
It also indicates that possibly degradation of this important cell cycle
regulator occurs at the fusome.

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Fig. 4. Expression of Cul1 in the Drosophila germarium. (A-C) The Cul1
protein (red) is a nuclear protein but in the wild-type germarium mostly
associates with the spectrosome (arrow) and the fusome (stained with
antibodies against the Hts protein, green). (D-F) In encore mutant
germaria, Cul1 association with the fusome is much-reduced (inset) and the
Cul1 staining is prominently nuclear. Wild-type and encore mutant
flies were raised at 29°C. (G) Genetic interactions between
encore and cul1, UbcD2 and effete alleles.
Reduction of cul1 gene dose in an encore mutant background
enhances the 32-cell egg chamber phenotype from 27 to 65% at 25°C and from
3 to 44% at room temperature (RT). Enhancement of the phenotype from 27 to 60%
at 25°C and from 3 to 17% at room temperature is also seen by reducing the
gene dose of UbcD2. More dramatic enhancement from 27 to 82% at
25°C and from 3 to 75% at room temperature is observed when
effete gene dose is reduced. Similar results were obtained using
different cul1 and effete alleles.
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Encore associates with components of the SCF-proteasome degradation
system
At higher temperatures, the rate of the ubiquitin-proteasome dependent
proteolysis decreases (Kuckelkorn et al.,
2000
) and polyubiquitinated proteins may require the activity of
ancillary proteins for proper substrate presentation and destruction by the
proteasome (Bercovich et al.,
1997
; Hohfeld et al.,
2001
; Wiederkehr et al.,
2002
). Our results indicate that Encore has a role in facilitating
the destruction of Cyclin E and suggest a possible physical interaction
between Encore, Cyclin E and components of the SCF pathway. We performed
immunoprecipitation assays followed by western blots using antibodies directed
against the Drosophila Cyclin E, Encore, Cul1 and against the
mammalian proteasome 19S-RP subunit S1 and the proteasome 20S-CP subunit LMP7.
We found that Encore can immunoprecipitate Cyclin E and that antibodies
against Ubiquitin recognize this protein
(Fig. 5A). Unexpectedly,
antibodies against Encore can immunoprecipitate Cyclin E in encore
mutant extracts. These immunoprecipitation experiments were performed using
the point mutant allele encQ4 and the P-element insertion
allele encBB. Both encore mutant alleles produce
protein (not shown). Encore and Cul1 antibodies can immunoprecipitate the same
Cyclin E-Ubn and there is more Cyclin E-Ubn
immunoprecipitated in the encore lanes compared with the wild-type
lanes. Significantly, Cyclin E and Cul1 can associate with Encore
(Fig. 5B). These observations
suggest that Cyclin E-Ubn, Cul1 and Encore can form a complex. By
contrast, the anti-Encore antibody did not precipitate Fizzy, Cyclin A or B
(data not shown). Antibodies against the S1 and LMP7 proteins
immunoprecipitate several Cyclin E-Ubn forms of the same (5A longer
exposure) and higher molecular weight than the Cyclin E-Ubn
associated with Cul1 and Encore. LMP7 seems to bring down more Cyclin
E-Ubn in wild-type extracts compared with the encore
mutant extracts. Antibodies against S1 and LMP7 can immunoprecipitate several
Encore forms, suggesting that Encore may be part of a complex formed by the
SCF-proteasome system and that Encore may be a substrate for UPS
(Fig. 5B). The fact that Encore
can be seen in the mutant and wild-type lanes suggests that the defective
protein can still form a complex with SCF-proteasome components. At the moment
we do not have a protein null encore mutation and therefore we do not
know whether Encore is required for complex formation in the germarium.

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Fig. 5. Encore associates with Cyclin E, Cul1, the proteasome 19S-RP subunit S1 and
the proteasome 20S-CP LMP7. (A) Immunoblot (IB) using antibodies against
Cyclin E and Ubiquitin showing that Cul1 and Encore can immunoprecipitate
Cyclin E-Ubn. Immunoprecipitation with S1 and LMP7 precipitates
several polyubiquitinated Cyclin E forms. The control lane (co) refers to
immunoprecipitations that use unrelated antibodies raised in the same animal
as the antibody used for the immunoprecipitation test. (B) IB using antibodies
against Encore shows that Cyclin E, Cul1, S1 and LMP7 associate with Encore in
the ovary. Notice that the amounts of immunoprecipitated protein in the Cul1
panel are comparable in wild-type and encore mutant extracts. The S1
and LMP7 immunoprecipitation lanes show that several forms of Encore
associates with S1 and LMP7. The wild-type extract lane indicates the position
of Encore in the gel. The extracts were run in 7%Tris-Acetate Nupage gel
(Invitrogen).
|
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The proteasome 19S subunit S1 expression is defective in
encore mutant germaria
The localization of Cul1 to the fusome suggests that perhaps Cyclin E and
other SCF-UPS substrates may be degraded at the fusome. To test whether the
proteasome is also localized to the fusome, immunostaining assays were
performed using antibodies against the proteasome 19S-RP subunit S1. Indeed
the 19S-S1 colocalizes with the fusome in wild-type germaria
(Fig. 6A-C). 19S-S1 association
with the fusome is incomplete (arrowheads), as not all the fusomes are
associated with S1. Unlike Cul1, S1 seems to be associated only with some
areas of the fusome. Moreover there is also S1 accumulation in a granular
appearance in the rest of the germarium. In encore mutant ovaries of
flies raised at the restrictive temperature of 29°C, S1 expression is very
much reduced (Fig. 6D-F).
However some S1 protein can still be seen localized to the fusome. Thus, it
seems that Cul1, 19S-S1 and presumably the rest of the 19S-RP associates with
the fusome. The strong reduction of Cul1 localization to the fusome in
encore mutant germaria could result in inefficient recruitment of the
19S-RP and the rest of the proteolytic machinery.

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Fig. 6. Expression of the 19S-RP subunit S1 and P-Cyclin E in the
Drosophila germarium. (A-C) 19S-S1 (red) localizes to the fusome
(green, stained with Hts). Some fusomes are free of 19S-S1 staining
(arrowhead). S1 is also accumulated in a granular manner in the rest of the
germarium. (D-F) 19S-S1 expression in encore mutants is reduced;
however, some S1 protein can still by observed in the mutant germarium. (G-I)
In wild-type germaria, P-Cyclin E (red) localizes to the fusome (green). The
expression is transient as some fusomes express some P-Cyclin E and others do
not. (J-L) In encore mutant germaria, P-Cyclin E is more often
observed localized to the fusome.
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Phosphorylated Cyclin E association with the fusome is defective in
encore mutant germaria
Cyclin E/Cdk2 activity is regulated by auto-phosphorylation of its
regulatory subunit, Cyclin E (Clurman et
al., 1996
). Phosphorylation of Cyclin E results in the disassembly
of the Cdk2/Cyclin E complexes, follow by ubiquitination and destruction via
the SCF-UPS. Our results predict that phosphorylated Cyclin E (P-Cyclin E)
would be degraded at the fusome. Using an anti-P-Cyclin E antibody we found
that P-Cyclin E is expressed at the tip of the wild-type germarium in region 1
and it associates with the fusome (Fig.
6G-I). Some cysts contain very high levels of P-Cyclin E that fill
the cyst completely, other cysts express intermediate levels or no P-Cyclin E.
Unlike expression of Cul1, P-Cyclin E association with the fusome is not
observed in all cysts suggesting some periodicity. In encore mutant
germaria of flies raised at 29°C, P-Cyclin E is present in most of the
fusomes observed in a given germarium (Fig.
6J-L). These observations suggest that in encore mutant
germaria, PCyclin E localization to the fusome occurs and because the
degradation process is inefficient, more P-Cyclin E accumulates at the
fusome.
The Ubiquitin-proteasome pathway requires Encore activity for proper
protein turnover
Our data shows that Cyclin E can be ubiquitinated in encore mutant
germaria and that the defect resides in the destruction of Cyclin
E-Ubn. Polyubiquitinated proteins are recognized by the proteasome
19S-RP, deubiquitinated by resident isopeptidases and unfolded before being
destroyed by the proteasome 20S-CP (Cope et
al., 2002
; Verma et al.,
2002
). In order to test whether encore mutations affect
the activity of the proteasome 20S-CP subunit, we measured the rate of
proteolysis in wild type and encore mutant germaria-enriched
extracts. The peptidase activity was monitored by the hydrolysis of the
fluorescent-labeled peptide Suc-LLVYMCA
(Glass et al., 1998
). The
results show that rate of proteolysis in encore mutant extract of
females raised at 29°C or room temperature is comparable to that of wild
type extracts of flies raised at 29°C
(Fig. 7A). These results
suggest that Encore does not affect the peptidase activity of the proteasome
and that the defect may reside in substrate recognition, the formation of an
inactive complex and/or in subcellular localization of the proteolysis
machinery. We also tested the requirement of Encore for proper proteolysis
using an exogenous mammalian protein (Fig.
7B). Commercially available histidine-tagged P27 was ubiquitinated
in vitro. The necessary E1, E2 and E3 enzymes for the ubiquitination reaction
were provided by germaria-enriched extracts. There was no difference in
ubiquitination efficiency between wild-type and encore mutant
extracts (Fig. 7B, left). Equal
number and amount of ubiquitinated p27 bands are observed in both extracts.
The P27-Ubn was then purified using nickel-agarose beads and a
deubiquitination reaction was performed using either wild-type or
encore mutant germaria-enriched extract to provide the proteolysis
machinery. Time zero is the P27-Ubn from the wild-type
ubiquitination reaction (Fig.
7B, left). Deubiquitination and destruction of p27 was compromised
when encore mutant extracts were used
(Fig. 7B, right). In the
wild-type situation, after 20 minutes of incubation most of the ubiquitinated
p27 had disappeared. By contrast, after 1 hour, ubiquitinated p27 can still be
detected in the reaction using encore mutant extracts.

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Fig. 7. Encore is required for proteolysis. (A) Proteasome assays measuring the
hydrolysis of the fluorogenic peptide Suc-LLVYMCA. The 20S-CP proteolysis
activity is not significantly compromised in encore mutant
germarium-enriched extracts at 29°C (yellow) or room temperature (light
blue) compared with wild-type extracts at 29°C (pink). Control assays
contain no substrate peptide (dark blue). (B) Western blot using anti p27
antibodies show that Ubiquitination reactions produce the same
polyubiquitinated p27 forms (arrowheads) in wild-type and encore
mutant extracts. The deubiquitination and degradation reaction is much slower
when encore mutant extract was used as a source of the degradation
machinery. (C) Model of Encore function. Cul1 (pink) is localized to the
fusome (green) in an Encore (blue)-dependent manner (Encore may directly or
indirectly modify Cul1 and thus influence its subcellular localization). Cul1
may serve as an anchor where the SCF E3 complex is assembled and a
phosphorylated substrate (yellow) is recognized and ubiquitinated. The
polyubiquitinated substrate is then recognized by the 19S-recognition
particle. 19S-RP and presumably the 20S-core particle are recruited to the
fusome where the substrate is de-ubiquitinated, unfolded and degraded by the
26S-subunit of the proteasome.
|
|
 |
Discussion
|
---|
Cyclin E proteolysis and exit of mitosis in the Drosophila
germarium
Cell cycle transition is mainly driven by Cdk activity, which is carefully
regulated by the levels of the cyclin subunits, by CKI and by Cdk
post-translational modifications. Presumably, cyclin proteolysis is important
for ensuring the sharp decrease in Cyclin E expression after the fourth
mitosis which allows exit from the cell division program. Our genetic,
biochemical and immunostaining results indicate that in encore mutant
germaria Cyclin E proteolysis is defective which creates a surplus of Cyclin E
that induces the cysts to undergo an extra cell division. Much of the
accumulated Cyclin E is polyubiquitinated. Cyclin A also accumulates in
encore mutant germaria possibly in part as a result of defective
Cyclin E proteolysis. Reduction of Cyclin A activity can partially suppress
the encore extra mitosis phenotype. We think it likely that the extra
cell division is due to the combined effect of surplus Cyclin E, Cyclin A and
other proteins such as Bam (Hawkins et
al., 1996
). encore mutant ovaries containing additional
mutations in genes coding for the ubiquitin-proteasome components such as
Cul1, effete and UbcD2 show enhancement of the extra
division phenotype. We believe that this is the result of slower destruction
of Cyclin E and consequent protein accumulation. The fact that the extra
division phenotype can be observed at room temperature in these double mutants
suggests that Encore is involved in the proper destruction of Cyclin E and
perhaps other proteins degraded by the SCF pathway. Lilly et al.
(Lilly et al., 2000
) showed
that mutations in the E2 UbcD1 gene produce egg chambers with an
extra round of mitosis and that reducing Cyclin A and Cyclin
B but not Cyclin E gene dose suppresses the extra division
phenotype. It therefore appears that reducing Cyclin E dose in an
UdcD1 mutant background still leaves enough active Cyclin E to
promote an extra mitosis, whereas in the encore mutant background the
increased levels of active Cyclin E may be closer to a threshold.
The fusome is a regulator of cell division during early
oogenesis
Some of the functions ascribed to the fusome are to synchronize cyst
mitosis and to provide the scaffold for the transport system necessary for
oocyte determination (Lin et al.,
1994
; McGrail and Hays,
1997
; Deng and Lin,
1997
). Limiting the number of cell divisions in the germarium
could be achieved by regulating the association of proteins such as the
cyclins and/or other cell cycle regulators with the fusome. The expression
pattern of Cyclin A, Cul1, P-Cyclin E and 19S-S1 proteins in the germarium
supports the idea that the fusome plays an important role in the regulation of
mitosis. Indeed, Cyclin A association with the fusome is transient and occurs
only during cyst division (Lilly et al.,
2000
). In encore mutant germaria, Cyclin A remains
associated with the fusome after cell division has stopped. Cul1 localization
to the fusome suggests that the rest of the SCF complex also associates with
the fusome and that substrate ubiquitination may happen at the fusome
(Fig. 7C). We have shown that
the SCF component Cul1 is mainly associated with the fusome in the wild-type
germaria. In encore mutant germaria, Cul1 localization to the fusome
is very poor, leading us to propose that this may be one reason why Cyclin E
is not degraded properly. This also suggests that the degradation of Cyclin E
and perhaps of other proteins degraded by the SCF-UPS may occur at the fusome.
The association of P-Cyclin E supports this idea. The localization of P-Cyclin
E in the wild type seems to be dynamic, consistent with the idea that the
phosphorylated substrate is localized to the fusome, and then rapidly degraded
via the SCF-UPS. In encore mutant germaria, the poor localization of
Cul1 may result in an inefficient assembly of SCF complexes at the fusome.
P-Cyclin E is localized to the fusome, but its degradation is compromised and
as a result we observed a consistent expression of P-Cyclin E at the fusome.
The partial association of the proteasome 19S-RP subunit S1 to the fusome
supports the idea that proteolysis may occur at the fusome. The proteasome
19S-RP would recognize the polyubiquitinated substrate and recruit the rest of
the proteasome to the fusome (Fig.
7C).
The role of Encore on facilitating Cyclin E proteolysis
Our results suggest that Encore can associate with the SCFUPS machinery and
assists with the degradation of Cyclin E and perhaps other SCF substrates. As
the mutant Encore protein can still interact with SCF-UPS components, the
mutant protein may form complexes but these might be inactive and/or the
mutant protein poisons the degradation machinery. Consistent with such a
hypothesis, the encore extra cell division phenotype is milder in
hemizygous versus homozygous females at 25°C
(Hawkins et al., 1996
). Encore
is required for the proper localization of Cul1, P-Cyclin E, S1 and presumably
the rest of the proteolysis complex to the fusome. This localization may be
more crucial at 29°C, whereas at lower temperatures a less efficient
degradation system may have enough time for normal cell cycle regulation.
encore mutations do not affect the 20S-Core Particle activity as
measured by the rate of degradation of a fluorogenic peptide. We do not know
whether Encore retains Cul1 at the fusome or whether Encore directly or
indirectly modifies Cul1 in order to promote its localization at the fusome.
Cul1 is known to be modified by the addition of Nedd8
(Furukawa et al., 2000
);
however, Cul1 seems to be equally neddylated in encore and wild-type
ovary extracts (data not shown).
In summary, our results suggest that the Encore protein assists with proper
cell cycle progression in the Drosophila germarium by ensuring that
Cul1 and the proteolysis machinery is localized at the mitosis coordination
center, the fusome.
 |
ACKNOWLEDGMENTS
|
---|
We thank L. Cooley, J. de Nooij, V. Fillipov, C. Lehner, M. A. Lilly, D.
McKearin, H. Richardson and L. Zipursky for kindly providing us with stocks,
cDNA and antibodies. We also thank Joe Goodhouse for his help with the
confocal microscopy. We are grateful to Kristina Wehr and Andrew Swan for
comments on the manuscript, and the rest of the Schupbach laboratory for
suggestions and discussion. This work was supported by the Howard Hughes
Medical Institute and the NIH grant PO1 CA41086.
 |
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