3083Research Article

IntroductionThe ubiquitin-proteasome system is responsible for the controlled

intracellular degradation of short-lived or misfolded proteins. The

selective recognition of these proteins is ensured by members of

the ubiquitylation enzyme cascade. Ubiquitin-conjugating enzymes

and ubiquitin ligases, alone or in combination, are responsible for

substrate recognition. Through their catalytic activities, the selected

proteins are marked for intracellular degradation by the covalent

attachment of a polyubiquitin chain. Polyubiquitylated proteins are

selectively recognized and degraded by the 26S proteasome, a large

proteolytic complex that is assembled in an ATP-dependent reaction

from two distinct subcomplexes: the regulatory particle (RP) and

the catalytic particle (CP) (reviewed by DeMartino and Gillette,

2007). The CP is a nonspecific protease – it cannot discriminate

between polyubiquitylated and non-ubiquitylated proteins. The strict

specificity of the 26S proteasome for polyubiquitylated proteins,

which is absolutely essential for the maintenance of cellular

integrity, is ensured by the RP. Additionally, RPs are responsible

for unfolding the substrate proteins via their anti-chaperone activity,

opening the gated channel of the CP, reprocessing the ubiquitin

residues of the substrate proteins, and feeding the substrate into the

CP. The contributions of the individual RP subunits to the different

steps of the proteolytic process are far from clear.

The mechanism that ensures the selective recognition of the

polyubiquitin chain by the RPs is a crucial step in controlled

intracellular protein degradation, and has been studied in detail.

Two different classes of proteins have been identified that fulfil

all the criteria required for a polyubiquitin receptor. One of them,

the Rpn10/S5a/p54 protein (yeast/human/Drosophila orthologs),

can bind both polyubiquitylated substrate proteins and the

polyubiquitin chain with high affinity in different in vitro systems

(Deveraux et al., 1994; van Nocker et al., 1996; Haracska and

Udvardy, 1997; Fu et al., 1998). Because it is a stoichiometric

subunit of the RP, Rpn10/S5a/p54 was considered to be an obvious

candidate as the polyubiquitin receptor of the RP. Its role as a

polyubiquitin receptor, however, has been questioned in

consequence of the observation that deletion of the yeast Rpn10is not lethal, produces only mild phenotypic changes and does not

cause the accumulation of polyubiquitylated proteins in yeast cells

(van Nocker et al., 1996).

Recognition of polyubiquitylated substrates by the proteasomeis a highly regulated process that requires polyubiquitinreceptors. We show here that the concentrations of theproteasomal and extraproteasomal polyubiquitin receptorschange in a developmentally regulated fashion. Thestoichiometry of the proteasomal p54/Rpn10 polyubiquitinreceptor subunit, relative to that of other regulatory particle(RP) subunits falls suddenly at the end of embryogenesis,remains low throughout the larval stages, starts to increase againin the late third instar larvae and remains high in the pupae,adults and embryos. A similar developmentally regulatedfluctuation was observed in the concentrations of the Rad23and Dsk2 extraproteasomal polyubiquitin receptors. Depletionof the polyubiquitin receptors at the end of embryogenesis isdue to the emergence of a developmentally regulated selectiveproteolytic activity. To follow the fate of subunit p54/Rpn10 invivo, transgenic Drosophila melanogaster lines encoding the N-terminal half (NTH), the C-terminal half (CTH) or the full-length p54/Rpn10 subunit were established in the inducibleGal4-UAS system. The daughterless-Gal4-driven whole-bodyexpression of the full-length subunit or its NTH did not produceany detectable phenotypic changes, and the transgenic productswere incorporated into the 26S proteasome. The transgene-encoded CTH was not incorporated into the 26S proteasome,

caused third instar larval lethality and was found to be multi-ubiquitylated. This modification, however, did not appear to bea degradation signal because the half-life of the CTH was over48 hours. Accumulation of the CTH disturbed thedevelopmentally regulated changes in subunit composition ofthe RP and the emergence of the selective proteolytic activityresponsible for the depletion of the polyubiquitin receptors.Build-up of subunit p54/Rpn10 in the RP had already startedin 84-hour-old larvae and reached the full complementcharacteristic of the non-larval developmental stages at themiddle of the third instar larval stage, just before these larvaeperished. Similar shifts were observed in the concentrations ofthe Rad23 and Dsk2 polyubiquitin receptors. The postsyntheticmodification of CTH might be essential for this developmentalregulation, or it might regulate an essential extraproteasomalfunction(s) of subunit p54/Rpn10 that is disturbed by theexpression of an excess of CTH.

Supplementary material available online at

http://jcs.biologists.org/cgi/content/full/122/17/3083/DC1

Key words: Polyubiquitin receptors, 26S proteasome, Regulatory

particle, Multiubiquitylation

Summary

Developmental-stage-specific regulation of thepolyubiquitin receptors in Drosophila melanogasterZoltán Lipinszki1, Petra Kiss1, Margit Pál1, Péter Deák1, Áron Szabó1, Eva Hunyadi-Gulyas2, Eva Klement2,Katalin F. Medzihradszky2 and Andor Udvardy1,*1Institute of Biochemistry and 2Proteomics Research Group, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged,PO Box 521, Hungary*Author for correspondence (udvardy@brc.hu)

Accepted 3 June 2009Journal of Cell Science 122, 3083-3092 Published by The Company of Biologists 2009doi:10.1242/jcs.049049

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The discovery of a group of proteins (Rad23, Dsk2 and Ddi1)

containing ubiquitin-associated (UBA) and ubiquitin-like (UBL)

domains seemed to resolve this contradiction (reviewed by

Hartmann-Petersen and Gordon, 2004). The UBA of these proteins

ensures the selective recognition and binding of the polyubiquitin

chain, whereas the UBL, by its ability to bind to the RP, performs

the targeting of the polyubiquitylated proteins to the 26S

proteasome. Accumulation of polyubiquitylated proteins in rad23Δand dsk2Δ yeast mutants promoted the view that these non-

proteasomal proteins might function as polyubiquitin receptors

(Wilkinson et al., 2001). Following more detailed studies, however,

serious contradictions emerged concerning the exclusive role of

UBA-UBL proteins as polyubiquitin receptors. In rad23Δrpn10Δdouble-mutant yeasts, more pronounced pleiotropic defects were

observed than in either single mutant, and the canavanine-sensitivity

of the double-mutant strain was about 100-fold higher than that of

either single mutant (Lambertson et al., 1999; Elsasser et al., 2004;

Verma et al., 2004). This observation suggested that the

polyubiquitin receptor function requires some kind of cooperation

between the Rpn10 RP subunit and a UBA-UBL protein. In vitro

reconstitution of the 26S proteasome-dependent degradation of the

polyubiquitylated Sic1 (UbSic1) protein confirmed this hypothesis

(Verma et al., 2004). 26S proteasomes prepared from rad23Δ or

rpn10Δ yeast mutants failed to bind and degrade UbSic1.

Recombinant Rpn10 or Rad23 complemented the degradation

failure of the respective mutant proteasomes, but the

complementation of rad23Δ required the presence of at least the

N-terminal von Willebrand factor A (VWA) domain of Rpn10 in

the RP, in addition to Rad23. This observation clearly supported

the assumption that the interaction of Rpn10 with a UBA-UBL

protein is essential for the targeting and degradation of the

polyubiquitylated proteins. The physiological significance of these

in vitro studies was further confirmed by in vivo analysis of the

degradation of relevant proteasomal substrate proteins. These in vivo

studies demonstrated that an unexpected degree of specificity of

the polyubiquitin receptors was necessary for the in vivo degradation

of different polyubiquitylated proteins (Verma et al., 2004).

Analysis and comparison of the in vivo and in vitro data, however,

hint that alternative, hitherto-unresolved mechanisms might

complicate the events of the substrate recognition cycle. Thus, the

mechanism proposed for the recognition and targeting of

polyubiquitylated proteins does not explain the phenomenon that

the degradation of polyubiquitylated proteins is severely inhibited

in the presence of an excess of S5a (Deveraux et al., 1995) or Rpn10

polyubiquitin receptors (Verma et al., 2004). Furthermore, the

reversible dissociation-association observed in vitro for the yeast

(Babbitt et al., 2005) or Drosophila (Kiss et al., 2005) proteasomal

polyubiquitin receptor subunits (Rpn10 or p54, respectively), which

accompanies the in vitro assembly-disassembly process of the 26S

proteasome, indicates either that the recognition and targeting of

polyubiquitylated proteins is a more complex process, or that the

proteasomal polyubiquitin receptors have a yet-unknown

extraproteasomal function(s). The latter is supported by the

observation that, in both Drosophila (Haracska and Udvardy, 1995)

and yeast (van Nocker et al., 1996), some of the proteasomal

polyubiquitin receptors are found in a proteasome-unassociated

pool. The presumed extraproteasomal role of Rpn10 and its

extraproteasomal interaction with the Dsk2 polyubiquitin receptor

are supported by the in vivo observation that the severe cytotoxicity

of Dsk2 overexpression in yeast is mitigated by the accumulation

of extraproteasomal Rpn10 (Matiuhin et al., 2008). The CTH of

Rpn10, carrying the ubiquitin-interacting motif (UIM) is alone

responsible for this compensatory function, suggesting that in its

extraproteasomal state the UIM of Rpn10 might mask the UBL

domain of Dsk2, competing in this way with the interaction of Dsk2

with the proteasome. However, the presumed extraproteasomal

shuttling of the proteasomal polyubiquitin receptor is questioned

by the observation that human 26S proteasomes degraded

polyubiquitylated Sic1 and c-IAP1 proteins in vitro without

dissociating into the RP and CP and releasing any subunit

(Kriegenburg et al., 2008).

In the present work, the presumed shuttling cycle of the

Drosophila proteasomal polyubiquitin receptor subunit (p54) was

studied by exploiting the observation that the stoichiometry of the

RP subunits exhibits characteristic developmental changes. As

compared with embryos, there is a sudden drop in the relative

concentration of subunit p54 in the RP during the first instar larval

stage; the concentration remains low throughout the larval stages,

starts to increase at the end of the third instar larval stage, and again

reaches a high level in pupae and adults. To investigate the

regulatory events leading to the down-regulation of subunit p54

during the larval stages, transgenic Drosophila stocks were

generated to express different domains of subunit p54, and the

intracellular fates of both the transgenic proteins and the endogenous

p54 subunit were followed.

ResultsDevelopmental fate of subunit p54For an analysis of the fate of subunit p54 in synchronously

developing wild-type D. melanogaster, eggs were laid on agar plates

for 1 hour at 25°C and incubated at this temperature for various

times. Total protein extracts were prepared from animals in different

developmental stages, and the subunit compositions of the 26S

proteasomes were analyzed by an immunoblotting technique, using

a mixture of monoclonal antibodies recognizing the p48A (Rpt3)

ATPase subunit of the RP base, the p42A (Rpn7) and p39A (Rpn9)

subunits of the RP lid, three different monoclonal antibodies

recognizing three different epitopes (see below) located in the CTH

of subunit p54 (Rpn10), and a polyclonal antibody developed against

the CP. The loading of equal amounts of total proteins from the

different developmental stages (Fig. 1A) revealed that the

stoichiometry of subunit p54 dropped dramatically during the first

instar larval stage, remained low throughout the larval stages, started

to increase at the end of the third instar larval stage, and reached

the same high level in pupae and adults as is characteristic for the

early embryos (Fig. 1B).

Individually, all three anti-p54 monoclonal antibodies,

recognizing three different epitopes of the subunit, detected the

same developmental switch (data not shown). Thus, it is very

unlikely that a change of a postsynthetic modification recognized

by a monoclonal antibody is responsible for the developmental

switch. There was no detectable fluctuation in the stoichiometry

of other RP or CP subunits during the development of Drosophila.

Although a fraction of the p54 subunit is present in a proteasome-

unassociated pool in the ATP-depleted embryonic extract (Haracska

and Udvardy, 1995), the sudden drop in the p54 content of the RP

in the larvae is not due to the removal of this proteasome-

unassociated p54 pool during the embryo-larva developmental

transition, because only trace amounts of p54 protein were present

extraproteasomally in total embryonic protein extracts prepared in

the presence of ATP and fractionated on a Superose 6 sizing column

in ATP-containing buffer (supplementary material Fig. S1).

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Reduction of the concentration of subunit p54 during the larval

developmental stages might, in principle, be compensated by

overexpression of the non-proteasomal polyubiquitin receptors.

This is not the case, however, at least for the two major non-

proteasomal polyubiquitin receptors. As shown in Fig. 1C, there was

a serious reduction in the concentrations of the Rad23 and Dsk2

proteins during the larval stages, and the developmental profile of

this down-regulation was very similar to that observed for p54. This

suggests that the polyubiquitin receptors of the ubiquitin-proteasome

system are down-regulated during larval development. In contrast

to the serious reduction in the concentrations of the proteasomal and

extraproteasomal polyubiquitin receptors, there was no detectable

accumulation of the polyubiquitylated proteins during the early larval

stages (supplementary material Fig. S2), probably because of the

very low mitotic activity in the larvae. Our unpublished results have

revealed that two other yeast extraproteasomal polyubiquitin receptor

orthologs, the UBA-UBL protein Ddi1 [CG4420] and the Pru domain

containing Rpn13 [CG13349, p42E], have only weak affinity for

polyubiquitylated proteins in D. melanogaster (Z.L., M.P., P.D. and

A.U., unpublished results). For this reason, only the two major known

polyubiquitin receptors were examined in this work.

To test whether transcriptional regulation of the genes encoding

the individual RP subunits is responsible for the depletion of subunit

p54 in the larvae, total RNA was prepared from all the

developmental stages analyzed above, and the changes in the relative

concentrations of the mRNAs encoding the p54 and p48B subunits

were tested by a semi-quantitative RT-PCR technique. There was

no detectable drop in the level of the p54/p48B mRNA ratio in the

first instar larvae as compared to embryos, and this ratio seemed

to remain constant during the later larval stages. The p54/p48B

mRNA ratio increased slightly in the pupae and adults (Fig. 1D).

This observation clearly indicates that the depletion of subunit p54

in first instar larvae is regulated either at the level of p54 mRNA

translation, or by a selective proteolytic degradation of the p54

protein. Our results support this latter assumption.

The selectivity of the postulated proteolytic degradation should

ensure the elimination of a limited set of proteins, exclusively during

the early larval developmental stages. To test this assumption, an

aliquot of total embryonic protein extract was incubated at 25°C

for 10, 30 or 60 minutes with total protein extracts prepared from

synchronized 60-, 72-, 84-, 96-, 108- or 120-hour-old larvae.

Embryonic protein extract incubated at 25°C for 60 minutes without

Fig. 1. Developmental changes in the subunit composition of the RP. (A) Equal amounts of total proteins prepared from different developmental stages of wild-typeD. melanogaster were fractionated on 9% SDS-polyacrylamide gel and analyzed by silver staining. (B) The same amount of proteins were analyzed byimmunoblotting with a mixture of monoclonal antibodies recognizing p54, p48A, p42A and p39A RP subunits, and with a polyclonal antibody developed againstthe CP. (C) Lanes 1-15. Immunoblot analysis with same amount of protein samples described above using a mixture of polyclonal anti-Rad23 and anti-Dsk2antibodies. Lane 16: total embryonic protein extract reacted only with anti-Rad23 antibody. Lane 17: total embryonic protein extract reacted only with anti-Dsk2antibody. (D) Semi-quantitative RT-PCR analysis of p54 and p48B mRNAs in different developmental stages of D. melanogaster. rpL17A ribosomal proteinmRNA served as loading control.

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any larval extract served as control. The reactions were terminated

by boiling the samples in SDS sample buffer, and the changes in

the subunit pattern of the 26S proteasomes were analyzed using an

immunoblotting technique. In these reaction mixtures, the

embryonic 26S proteasome molecules provided most of the p54

subunit. As shown in Fig. 2A, protein extracts derived from 60-,

72-, 84- and 96-hour-old larvae selectively degraded the p54

subunit, leaving all the other proteasomal subunits intact. The

selectivity of this proteolytic degradation was supported by the

observations that glycogen phosphorylase and β-importin proteins

(present in both the embryonic and the larval protein extracts)

remained completely intact during this incubation, and no detectable

changes were noted in the silver-stained total protein profile of the

incubated samples (Fig. 2B). This is in sharp contrast to the selective

degradation of Dsk2 and Rad23 polyubiquitin receptors in early

larval protein extracts (Fig. 2C). The proteasome inhibitor MG132

did not inhibit the selective degradation of p54, Dsk2 and Rad23

by the larval extracts (data not shown).

Analysis of the in vitro degradation of subunit p54 by second

instar larval protein extracts revealed another level of selectivity.

In the immunoblotting experiments illustrated in Fig. 1B and Fig.

2A, the p54 subunit was detected by three monoclonal antibodies

that recognize the CTH of the subunit. To analyze the fate of the

NTH that carries the VWA domain of subunit p54 during the process

of in vitro degradation, the samples analyzed in Fig. 2A were re-

probed with a polyclonal antibody developed against recombinant

NTH of p54. A typical experiment with the 60-hour-old larval

extract demonstrates that, in vitro, the N-terminal fragment of the

proteasome-bound subunit p54 is highly protected against

proteolytic attack (Fig. 2D). The size of the protected polypeptide

is about 28 kDa, i.e. the whole VWA domain is protected. To follow

the physiological relevance of this observation, the protein samples

of synchronously developing wild-type D. melanogaster (presented

in Fig. 1B) were re-probed with the NTH-specific polyclonal

antibody (Fig. 2E). The protected polypeptide fragment detected in

the in vitro degradation assay was present in all the larval

developmental stages in which the full-length p54 was substantially

reduced, whereas the truncated subunit was undetectable in embryos,

pupae or adults. The truncated N-terminal fragment co-eluted with

the larval 26S proteasomes following size fractionation on Superose

6, indicating that the fragment is an integral component of the

proteasome (data not shown).

Ectopic expression of different domains of subunit p54For our study of the regulatory events leading to the down-

regulation of subunit p54 in the larvae, transgenic Drosophila stocks

were generated to express different domains of subunit p54, and

the fate, the postsynthetic modification and the phenotypic effect

of the ectopic expression of the transgenes were studied.

Transgenic constructs encoding the untagged or Strep-tagged

versions of the CTH, the NTH and the full-length p54 were

generated in the pP{UAST} vector. The NTH (1-613 bp of the

cDNA) carries the VWA domain required for the incorporation of

the subunit into the RP, whereas the CTH (614-1188 bp of the

cDNA) carries the UIM motifs responsible for the recognition and

binding of the polyubiquitin chain (Fu et al., 1998). Following

transformation, homozygous viable and fertile transgenic stocks

without any detectable abnormalities were selected. Transgene

expression was induced by crossing the transgenic flies to the Gal4

driver stock. This driver ensures a high-level expression of UAS-

Journal of Cell Science 122 (17)

Fig. 2. Selective proteolytic degradation of thepolyubiquitin receptors in the larvae of Drosophila.(A) Total embryonic protein extract was incubated at 25°Cfor the time indicated with an equal amount (by proteincontent) of larval protein extracts prepared fromsynchronized Drosophila larvae. Degradation of theproteasomal subunits was analyzed by immunoblottingtechnique using anti-p54, anti-p48A, anti-p42A, anti-p39Aand anti-CPα7 monoclonal antibodies. (B) Protein samplesdigested with 60-hour-old larval protein extract wereanalyzed by silver staining and immunoblotted with anti-glycogen phosphorylase and anti-β-importin antibodies.(C) Protein samples shown in A were analyzed byimmunoblotting with a mixture of anti-Dsk2 and anti-Rad23 antibodies. (D) Total embryonic protein extract wasincubated at 25°C for the time indicated with an equalamount (by protein content) of a larval protein extractderived from 72-hour-old synchronized larvae. The in vitrodegradation of subunit p54 was followed byimmunoblotting technique using a polyclonal antibodydeveloped against recombinant NTH of subunit p54.(E) Protein samples prepared from different developmentalstages of synchronized wild-type D. melanogaster (samplesof Fig. 1B) were analyzed by immunoblotting techniqueusing a polyclonal antibody developed against recombinantNTH of subunit p54.

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regulated transgenes in all cells of the transgenic animals during

all developmental stages.

Four different transgenic lines carrying the Strep-tagged full-

length p54 gene were crossed to daughterless-Gal4 (da-Gal4) and

the viability, fertility and appearance of visible morphological

abnormalities in the offspring analyzed. Expression of the Strep-

tagged full-length p54 did not cause any detectable changes in the

parameters tested. The eight-amino-acid-long Strep-tag allowed the

discrimination and simultaneous immunodetection of the

endogenous and transgenic p54 proteins. As shown in Fig. 3B,C,

the Strep-tagged full-length p54 protein was expressed when

induced by the da-Gal4 driver. The whole amount of the transgenic

product was incorporated into the 26S proteasome: after size-

fractionation of a total protein extract of da-Gal4/Strep-p54(7) stock,

all the Strep-tagged transgenic protein co-migrated with the

endogenous p54, the p42C (Rpt6) RP base and the p39 (Rpn9) RP

lid subunits, and no free transgenic protein was detected in the

monomeric protein fractions. Similar results were obtained with the

other three transgenic stocks expressing the Strep-tagged full-length

p54 (data not shown). Transgenic stocks expressing the NTH were

also viable and fertile, which is in contrast to the fact that the

truncated subunit was incorporated into the 26S proteasome (Fig.

3D). These findings are in sharp contrast to the phenotypic changes

observed following the expression of the CTH.

Four different transgenic lines carrying the 3�-half of the p54

gene were tested. Crossing each of these stocks to the da-Gal4 driver

resulted in third instar larval lethality. Transgene expression was

analyzed by an immunoblotting technique. Transgene-encoded

CTH protein was not detected in the transgenic animals before they

were crossed to the Gal4 driver, but induction with the driver resulted

in the accumulation of large amounts of CTH protein (Fig. 4). As

expected, the transgenic CTH protein lacking the VWA domain was

not incorporated into the 26S proteasome and appeared as a

monomeric protein after Superose 6 fractionation (Fig. 3E).

Transgenic line da-Gal4/C18, expressing the CTH at the highest

level, was chosen for further studies.

It was previously shown that an excess of S5a or Rpn10 inhibited

the proteasomal degradation of two different polyubiquitylated

proteins in vitro (Deveraux et al., 1995; Verma et al., 2004). Thus,

the lethal phenotype of CTH overexpression might be due to a severe

inhibition of the in vivo turnover of polyubiquitylated proteins. To

test this assumption, we analyzed the accumulation of

polyubiquitylated proteins in larvae following the induction of CTH

expression. As shown in Fig. 5, induction of the CTH resulted in

the accumulation of polyubiquitylated proteins in da-Gal4/C18

larvae.

Postsynthetic modification of the CTHA previous study of the Zn2+-induced reversible dissociation of

subunit p54 demonstrated that, during its extraproteasomal state,

this subunit interacts with several non-proteasomal proteins,

including the Smt3 SUMO-activating enzyme (Kiss et al., 2005).

The in vivo relevance of this interaction was confirmed by yeast

two-hybrid analysis: the NTH of p54 displayed a strong interaction

with the Smt3 SUMO-activating enzyme and the Ubc9 SUMO-

conjugating enzyme. This observation raised the possibility that,

during its presumed extraproteasomal state, this subunit becomes

postsynthetically modified, which might be important for an earlier

unrecognized extraproteasomal function of the subunit. As the CTH

cannot incorporate into the RP, its expression might model an

extended extraproteasomal state of the CTH of subunit p54,

allowing the formation and, hence, the accumulation and detection

of a presumed postsynthetic modification(s).

To study the occurrence of postsynthetic modification, a

transgenic Drosophila stock (8SC) carrying the Strep-tagged CTH

Fig. 3. Incorporation of transgene-encoded derivatives of subunit p54 into theRP. (A) Domain structure of subunit p54 and its transgenic derivatives.(B) Total protein extract prepared from Strep-p54(7) flies was fractionated on aSuperose 6 gel filtration column. The column fractions were separated on 8%SDS-polyacrylamide gel and tested by immunoblot analysis using a mixture ofmonoclonal antibodies specific for subunits p54, p42C (belonging to the RPbase) and p39A (belonging to the RP lid). (C) Total protein extract preparedfrom da-Gal4/Strep-p54(7) flies was fractionated on Superose 6 gel filtrationcolumn. The column fractions were tested as described in B. (D) Total proteinextract prepared from da-Gal4/Strep-NTH flies was fractionated on Superose 6gel filtration column. The column fractions were separated on 12% SDS-polyacrylamide gel and tested by immunoblot analysis using a polyclonalantibody that recognizes the NTH of subunit p54. The band marked by asterisk(*) in the monomeric protein fractions is a polypeptide nonspecificallyrecognized by the polyclonal antibody, with mass higher than that of the NTH.(E) Total protein extract prepared from heat-shocked R80/da-Gal4/C18 flieswas fractionated on a Superose 6 gel filtration column. The column fractionswere separated on 10% SDS-polyacrylamide gel and tested by immunoblotanalysis with p54 subunit-specific monoclonal antibodies.

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transgene was crossed to the da-Gal4 driver. The total protein extract

was prepared from da-Gal4/8SC third instar larvae and the Strep-

CTH protein was purified on a Strep-Tactin affinity column. The

affinity-purified proteins were fractionated on 10% SDS-

polyacrylamide gel and immunoblotted with anti-p54 monoclonal

antibodies. Total protein extract prepared from non-induced 8SC

larvae (i.e. before crossing to the da-Gal4 driver) served as control.

As shown in Fig. 6, a set of anti-p54 reactive proteins appeared in

the affinity-purified fraction derived from the larvae expressing the

Strep-tagged CTH. These proteins were completely missing in the

eluted fractions purified from non-induced larvae. The most

prominent of these bands, which was about 8 kDa larger than the

Strep-tagged CTH, was detectable even in the total larval protein

extract (see also Fig. 4). The band marked by an asterisk (Fig. 6)

corresponds to a nonspecifically bound full-length p54 (control

experiments with recombinant p54 proved that Strep-Tactin column

can nonspecifically bind p54, data not shown). The immunoreactive

bands formed a ladder, reminiscent of an ubiquitylation ladder.

To prove the presumed ubiquitylation, the affinity-purified

protein fraction was further purified by two-dimensional IEF-SDS

PAGE, exploiting the very acidic nature of the CTH (calculated pI

is 3.6). Two parallel two-dimensional gels were prepared: one of

them was silver-stained, and the other one was immunoblotted with

anti-p54 monoclonal antibodies to identify the CTH and its

derivatives (Fig. 7). The silver-stained spots marked in Fig. 7B were

cut out and analyzed by mass spectrometry. In addition to CTH-

derived peptides, ubiquitin-derived peptides were detected in all

the spots (supplementary material Fig. S3), providing direct

evidence for the in vivo ubiquitylation of the CTH. Our efforts to

identify the lysine residue(s) ubiquitylated in the CTH failed, due

to the low quantity of purified material recovered. Ubiquitylation

of the CTH can likewise be achieved in vitro by incubating the

recombinant CTH in an embryonic protein extract in the presence

of ATP and ubiquitin. The modified proteins were affinity-purified

and analyzed by immunoblotting technique (supplementary material

Fig. S4).

Ubiquitylation of the CTH is not a degradation signalIn vivo ubiquitylation of the yeast Rpn10 subunit has recently been

demonstrated (Crosas et al., 2006), though the site of the

ubiquitylation was not identified. The short half-life of the

ubiquitylated Rpn10 (45 minutes) suggested that a bona fide

polyubiquitin chain, a true degradation signal, is formed on the

Rpn10 subunit. With the aid of the heat-inducible driver, we

determined the half-life of the CTH protein. In this experiment,

R80/da-Gal4/C18 flies grown at 19°C were heat-shocked at 30°C

for 36 hours, and the incubation was then continued at 19°C for a

further 4 days. Total protein extracts were prepared from non-heat-

shocked and heat-shocked animals, and from animals kept for 1-4

days at 19°C following the heat-shock.

Immunoblot analysis (Fig. 8) revealed that at 19°C the CTH is

not expressed at all. Following 36 hours of heat shock, a large

amount of the CTH had accumulated. It proved to be a stable protein

as its half-life at 19°C was over 48 hours. Thus, the in vivo

ubiquitylation of the CTH does not serve as a degradation signal,

but is more probably connected with an essential extraproteasomal

function of subunit p54.

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Fig. 4. Driver-induced expression of the CTH transgene. Total protein extractsof third instar C18 larvae (lane 1) and third instar da-Gal4/C18 larvae werefractionated on 9% SDS-polyacrylamide gel and analyzed by immunoblottingwith p54 subunit-specific monoclonal antibodies.

Fig. 5. Accumulation of polyubiquitylated proteins in da-Gal4/C18 transgeniclarvae. Total protein extracts prepared from 84-hour-old (lane 1); 96-hour-old(lane 2); 108-hour-old (lane 3) and 120-hour-old (lane 4) C18 or from 84-hour-old (lane 5); 96-hour-old (lane 6); 108-hour-old (lane 7) and 120-hour-old(lane 8) da-Gal4/C18 larvae were fractionated on 7% SDS-PAGE and testedby immunoblotting technique using a mixture of anti-Ubiquitin and anti-glycogen phosphorylase antibodies. Glycogen phosphorylase (marked byarrow) was used as a loading control.

Fig. 6. Affinity purification of Strep-tagged CTH derivatives. Total proteinextract of da-Gal4/8SC third instar larvae (lane 1), proteins of the da-Gal4/8SCextract which flow through the Strep-Tactin column (lane 2) and proteinsaffinity-purified from da-Gal4/8SC protein extract on Strep-Tactin affinitycolumn (lane 3) was fractionated on 10% SDS-polyacrylamide gel and testedby immunoblot technique using anti-p54 monoclonal antibodies. Total proteinextract of non-induced 8SC third instar larvae (lane 4), proteins of the 8SCextract which flow through the Strep-Tactin column (lane 5) and proteinsaffinity-purified from 8SC protein extract on Strep-Tactin affinity column(lane 6) served as control. The asterisk marks the nonspecifically boundendogenous p54 protein.

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Ectopic expression of the CTH disturbs the developmentalregulation of polyubiquitin receptorsTo investigate the consequences of high-level CTH accumulation

on the developmental regulation of the p54 subunit in larvae, total

protein extracts were prepared from synchronously developing C18

and da-Gal4/C18 larvae, and the subunit composition of the 26S

proteasome was analyzed by immunoblotting with a mixture of

monoclonal antibodies as described above. In C18 larvae, as in wild-

type animals, the p54 subunit was present at low molar concentration

relative to other RP subunits during the early larval stages, and had

started to accumulate in 108-hour-old larvae during the second half

of the third instar larval stage (Fig. 9).

There are significant changes in the developmental regulation of

p54 subunit accumulation in da-Gal4/C18 larvae. The accumulation

of full-length p54 subunit has already started in 84-hour-old larvae,

and reaches the maximal level in 108-hour-old larvae, just before

these larvae start to perish. Similar results were observed with the

extraproteasomal polyubiquitin receptors (Fig. 9). In the C18 stock,

the concentrations of Rad23 and Dsk2 were low in the early larval

stages, and started to accumulate in the middle of the third instar

larval stage. In da-Gal4/C18 larvae, the accumulation of the

extraproteasomal polyubiquitin receptors started during the second

larval stage and reached its maximal level in the early third instar

larvae.

The data in Fig. 2 indicate that depletion of the polyubiquitin

receptors during the early larval developmental stages is a

consequence of the emergence of selective proteolytic activity. It

was reasonable to suppose that the disturbance of physiological

regulation observed following the ectopic expression of CTH (Fig.

9) might be a consequence of an alteration in the developmentally

regulated expression of this key proteolytic activity. To test this

assumption, a total embryonic protein extract was incubated at 25°C

for 15, 45 or 90 minutes with total protein extracts prepared from

synchronized 60-, 72-, 84-, 96-, 108- or 120-hour-old C18 or da-

Gal4/C18 larvae. Embryonic protein extract incubated at 25°C for

90 minutes without any added larval extract served as control. The

reactions were terminated by boiling the samples in SDS sample

buffer, and the changes in the subunit pattern of the 26S proteasomes

were analyzed by an immunoblotting technique, as described

above.

As shown in Fig. 10, the developmental profile of the selective

proteolytic activity of the synchronized C18 larval extracts was

indistinguishable from that of the wild-type larval extract. To detect

even weaker proteolytic activities, the in vitro incubation of

embryonic extract with larval extracts was extended for 15, 45 or

90 minutes in these experiments, and thus the p54 subunit of the

embryonic 26S proteasomes was completely degraded even during

the shortest incubation period. The ectopic expression of CTH in

the da-Gal/C18 larvae inhibited this selective proteolytic activity

in a developmentally regulated fashion. In early larval stages (60-

Fig. 7. Two-dimensional IEF-SDS PAGE fractionation ofaffinity-purified Strep-tagged CTH proteins. Affinity-purifiedStrep-tagged CTH protein derivatives shown in Fig. 6 wereseparated on pH 3–5.6 NL Immobilion DryStrip gel in the firstdimension and on 10% SDS-polyacrylamide gel in the seconddimension. Parallel gels were immunoblotted with anti-p54monoclonal antibodies (panel A) or silver stained for massspectrometry (panel B). Arrows indicate the spots analyzed bymass spectrometry.

Fig. 8. In vivo stability of the CTH protein. Total protein extracts of R80/da-Gal4/C18 flies kept at 19°C (lane 1), heat-shocked for 36 hours at 30°C (lane2), or grown for 24 hours (lane 3), 48 hours (lane 4), 72 hours (lane 5) or 96hours (lane 6) at 19°C following the 36 hours heat-shock were fractionated on10% SDS-polyacrylamide gel and immunoblotted with anti-p54 monoclonalantibodies.

Fig. 9. Accumulation of the transgenic CTH protein disturbs thedevelopmental regulation of the polyubiquitin receptors. (A) Total proteinextracts prepared from synchronously developing C18 and da-Gal4/C18 larvaewere fractionated on 9% SDS-polyacrylamide gel and analyzed byimmunoblotting. RP subunits were developed with the monoclonal antibodymixture described in Fig. 1; the CP was detected by a monoclonal antibodyspecific for the α7 subunit of the CP. Rad23 and Dsk2 proteins were detectedwith a mixture of the polyclonal anti-Rad23 and anti-Dsk2 antibodies.Glycogen phosphorylase used as a loading control was detected with anti-glycogen phosphorylase antibody.

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72 hours), the proteolytic activity was very strong, completely

degrading both the proteasome-bound full-length p54 polypeptide

and the ectopically expressed CTH. This proteolytic activity was

already significantly inhibited in 84-hour-old larvae and the

inhibition was complete in 96-hour-old larvae. The degradation of

Dsk2 and Rad23 was also inhibited in a similar developmentally

regulated fashion (data not shown).

These in vitro degradation data are congruent with the in vivo

changes of the proteasomal subunit patterns induced by the ectopic

expression of CTH (Fig. 9). All of the data presented indicate that

the accumulation of the CTH protein and/or its ubiquitylation

influence the normal developmental fate of the polyubiquitin

receptors in D. melanogaster by altering the developmentally

regulated expression of a selective proteolytic system.

DiscussionIn D. melanogaster, there is a developmentally regulated reduction

in the concentration of the proteasomal and extraproteasomal

polyubiquitin receptors during the larval stages. The physiological

relevance of this regulation is far from clear, but the very fast cell

division activity in the embryos, the highly active controlled

proteolysis required for the histolysis of larval structures during the

pupal stage, and the reproductive activity of the adults might explain

the need for a much higher polyubiquitin receptor concentration.

Our results demonstrate that a developmentally regulated selective

proteolytic activity is responsible for the changes in the

concentrations of the polyubiquitin receptors. This proteolytic

activity emerges in the early larvae and persists at a high level until

the middle of the third instar larval stage, gradually disappearing

thereafter.

The selectivity of this proteolytic activity displays several

unusual properties. It ensures the degradation of the proteasomal

and extraproteasomal polyubiquitin receptors, leaving the other

proteasomal subunits, the glycogen phophorylase and the β-importin

proteins intact. Furthermore, this proteolytic activity degrades only

the CTH of subunit p54 carrying the UIM motifs, but spares the

NTH of the subunit that interacts with the RP of the proteasomes

(Fu et al., 1998). The specificity is further supported by the

observation that, both in vivo and in vitro, this proteolytic activity

spares a well-defined fragment of subunit p54 of equal length.

Transgenic expression of the full-length p54 or its NTH does not

cause any detectable phenotypic changes in Drosophila, because

the cellular concentrations of these transgenic proteins are strictly

controlled. This follows from the observation that these transgenic

proteins do not accumulate in the monomeric, extraproteasomal

pool, the molar concentration of the endogenous and transgenic

proteins together corresponding to the level required for the

stoichiometric assembly of the RP (Fig. 3). The presence of the

VWA domain in the full-length transgenic p54 protein and in its

NTH ensures their incorporation into the RP. The excess transgenic

products are probably degraded, and the lack of unassembled

transgenic proteins might explain the viability and fertility of these

transgenic animals. The CTH cannot assemble into the RP due to

the lack of the VWA domain and, as it has a fairly long half-life,

the CTH accumulates in large quantity in the extraproteasomal pool.

All the in vitro and in vivo data so far published demonstrate

that any shift in the balance of the concentrations of the proteasomal

and extraproteasomal polyubiquitin receptors results in a serious

disturbance of the ubiquitin-proteasome system, which is always

manifested in the accumulation of polyubiquitylated proteins

(Deveraux et al., 1995; Ortolan et al., 2000; Kleijnen et al., 2000;

Raasi and Pickart, 2003; Verma et al., 2004; Matiuhin et al., 2008).

Although our data confirm these observations, we have noticed a

hitherto-unexpected consequence of the accumulation of the CTH

protein: the deregulation of the developmental program responsible

for adjustment of the concentrations of proteasomal and

extraproteasomal polyubiquitin receptors. RT-PCR analysis revealed

that the drop in the p54 content of the RP in the first instar larvae

is not regulated at the transcriptional level. It appears more probable

that, as in yeast, selective proteolytic degradation of p54 is

responsible for the developmental changes.

In yeast, subunit Rpn10 is a short-lived protein; it is

polyubiquitylated and degraded with a half-life of 45 minutes. The

half-life of Rpn10 is regulated by the competitive action of the hul5

Journal of Cell Science 122 (17)

Fig. 10. Accumulation of the transgenic CTH protein disturbs thedevelopmentally regulated expression of selective larvalproteolytic activity. (A) Total embryonic protein extract wasincubated at 25°C for the time indicated with an equal amount (byprotein content) of larval protein extracts prepared fromsynchronized C18 Drosophila larvae. Degradation of theproteasomal subunits was analyzed by immunoblotting techniqueas described in Fig. 2A. (B) Total embryonic protein extract wasincubated at 25°C for the time indicated with an equal amount (byprotein content) of larval protein extracts prepared fromsynchronized da-Gal4/C18 Drosophila larvae. Degradation of theproteasomal subunits was analyzed by immunoblotting techniqueas described in Fig. 2A.

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3091Developmental regulation of ubiquitin receptors

ubiquitin ligase, which promotes the polyubiquitylation of the

subunit, and the Ubp6 deubiquitylating enzyme, which removes the

polyubiquitin modification (Crosas et al., 2006). In Drosophila, at

least during the early larval stages, the fate of subunit p54 is

regulated by a different proteolytic mechanism. This follows from

the observation that the in vitro degradation of subunit p54 by early

larval protein extracts is resistant to the proteasome inhibitor

MG132.

Our in vitro studies (Z.L., M.P., P.D. and A.U., unpublished

results) reveal that the recombinant CTH can efficiently bind not

only the polyubiquitylated proteins, but also the Dsk2 and Rad23

extraproteasomal receptors. Thus, the ectopic accumulation of CTH,

which is a stable protein with a half-life of more than 48 hours,

might sequester both the polyubiquitylated proteins and the very

low amount of extraproteasomal polyubiquitin receptors present in

the larvae, in this way inhibiting the ubiquitin-proteasome system

of the larvae completely, and leading to death of the transgenic

animals.

Materials and MethodsThe cDNA-encoding subunit p54, and its NTH or CTH (1-613 and 614-1188 bp,

respectively) were cloned into the P-element based pP{UAST} vector. For

construction of the Strep-tagged versions of the transgenes, the cDNA derivatives

were first cloned into the pASK-IBA5 vector (IBA) in frame with the Strep-tag,

and the Strep-cDNA cassettes were inserted into the pP{UAST} vector. All the

constructs (Fig. 3A) were verified by DNA sequencing. The plasmid constructs

were injected into w1118 embryos, and transformed flies were selected according to

standard procedures (Spradling, 1986). Appropriate drivers of the Gal4 system

ensured the induction of transgene expression (Duffy, 2002). For construction of

the heat-inducible transgenic stock R80/da-Gal4/C18, flies with third chromosome

P-element insertions carrying the da-Gal4 driver and the CTH, and a second

chromosome P-element insertion carrying the heat-sensitive Gal 80ts repressor were

crossed. Gal 80ts reversibly inactivates the Gal4 transcription factor at 19°C.

Inactivation of the Gal 80ts repressor at 30°C allows the induction of transgene

expression.

To collect synchronously developing Drosophila samples, 200 virgin females from

the appropriate stocks (wild-type w1118 or C18 transgenic lines) were collected and

fed for 2 days on yeast-rich agar medium at 25°C. From the third day, they were

mated with 150 males (w1118 in the case of the wild-type; C18 or da-Gal4 in the case

of C18 control or da-Gal4/C18 induced stocks, respectively). Eggs were laid for 1

hour at 25°C. To obtain biological samples of different developmental stages, w1118

eggs were incubated at 25°C for 0-12 and 12-24 hours (embryos); 36 and 48 hours

(first instar larvae); 60 and 72 hours (second instar larvae); 84, 96, 108 and 120 hours

(third instar larvae); and till white puparium, brown puparium, pharate adult or newly

eclosed virgin female and male adult stages. In the case of C18 and da-Gal4/C18

stocks 84-, 96-, 108- and 120-hour-old larvae were collected. The synchrony of the

development and the developmental stages of Drosophila samples were checked by

appropriate morphological markers (Ashburner, 1989). The biological samples were

immediately frozen and stored at –80°C.

Protein extracts were prepared by homogenizing the synchronous Drosophilasamples for 1 minute at 4°C in 20 mM Tris-HCl at pH 7.6, 100 mM NaCl, 5 mM

MgCl2, 1 mM DTT, 1 mM ATP and 5% glycerol in a micro-Potter homogenizer. The

debris was removed by centrifugation at 14,500 g for 3 minutes at 4°C. The

supernatants were immediately mixed with one third volume of 4� SDS-sample buffer

and boiled for 4 minutes.

The cDNA encoding the Drosophila orthologs of the yeast Rad23 and Dsk2 UBA-

UBL proteins (CG1836 and CG14224: Ubiquilin, respectively) were PCR-amplified

with (Rad23 Fw: 5�-GCGCCTCGAGATTATTACAATTAAAAATCTT-3� and Rad23

Rev: 5�-GCGAGATCTCATTTTGTCATCCTAATC-3� or Dsk2 Fw: 5�-CTTAAG -

CTTATGGCGGAAGGCGGCAGCAA-3� and Dsk2 Rev: 5�-GGCTCGAGT TA -

ACTCAAGGACAACTGGTT-3�) primers, cloned into the pFLAG-MAC (Sigma

Aldrich) vector, and verified by DNA sequencing. Polyclonal anti-Rad23, anti-Dsk2

and anti-glycogen phosphorylase antibodies were raised in rabbits using FLAG-tagged

recombinant proteins expressed in E. coli and purified till homogeneity by the Anti-

FLAG M2 (Sigma Aldrich) affinity system (data not shown).

Native and denaturing protein gel electrophoresis, immunoblotting techniques, the

monoclonal antibodies, Superose 6 chromatography and mass spectrometric

techniques were as described earlier (Udvardy, 1993; Kiss et al., 2005). Anti-ubiquitin

antibody was purchased from Dako (Glostrup, Denmark). Strep-Tactin affinity

purification was carried out as suggested by the manufacturer (IBA, Göttingen,

Germany).

Total RNA was isolated with the Trizol reagent (Invitrogen) according to the

manufacturer’s protocol. After DNase treatment with RQ1 RNase-Free DNase

(Promega, Mannheim, Germany), samples were subjected to reverse transcription

using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania)

with oligodT primer. To compare the relative quantities of ribosomal protein L17A

cDNA (rpL17A, CG3661) in the different reverse transcribed samples, PCR was

performed with different amounts of cDNA samples using rpL17A specific primers

(rpL17A Fw: 5�-GTGATGAACTGTGCCGACAA-3� and rpL17A Rev: 5�-CCT -

TCATTTCGCCCTTGTTG-3�) and GoTaq (Promega) DNA polymerase for 25

cycles of amplification (95°C for 30 seconds, 60°C for 30 seconds, 72°C for 30

seconds). This PCR generates a 268-bp long fragment. PCR products were detected

in agarose gel and cDNA quantities giving the same band density among the different

samples were used to amplify the p54 and p48B cDNAs with (p54-1/3 Fw: 5�-CGAATTGCCCATCTGGTGCTGAA-3� and p54-2/3 Rev: 5�-TGCCGTTG CTC -

CTCCATAGACACA-3�or p48B-1/3 Fw: 5�-TAAGCAGTTCGCCAAGTTCG-3�and

p48B-2/3 Rev: 5�-CAGCACCTTGATATTTCCGC-3�) primers. This PCR generates

a 419-bp long p54 and a 610-bp long p48B fragment. After 25 cycles of amplification,

products were run on agarose gel and photographed.

Fly stocks were cultured at 25°C on standard Drosophila food. A w1118 isogenic

stock was used as wild type (Ryder et al., 2004). All genetic markers used are described

by Lindsley and Zimm (Lindsley and Zimm, 1992).

Genotypes of the Drosophila transgenic stocksC18: w1118; If/CyO; P{UAST-p54-CTH}8SC: w1118; If/CyO; P{UAST-Strep-p54-CTH}Strep-p54(7): w1118; If/CyO; P{UAST-Strep-p54}Strep-NTH: w1118; If/CyO; P{UAST-Strep-p54-NTH}da-Gal4: w1118; +; P{da-Gal4}da-Gal4/C18: w1118; CyO/+; P{da-Gal4}/ P{UAST-p54-CTH}da-Gal4/8SC: w1118; CyO/+; P{da-Gal4}/ P{UAST-Strep-p54-CTH}da-Gal4/Strep-p54(7): w1118; CyO/+; P{da-Gal4}/ P{UAST-Strep-p54}da-Gal4/Strep-NTH: w1118; CyO/+; P{da-Gal4}/ P{UAST-Strep-p54-NTH}R80: w1118; P{ tubP-Gal80ts}20; TM2/TM6R80/da-Gal4/C18: w1118; P{tubP-Gal80ts}20; P{UAST-p54-CTH},P{da-Gal4}

This work was supported by a grant from the Hungarian ScientificResearch Fund (OTKA T046177). This publication was supported bythe Dr Rollin D. Hotchkiss Foundation. The Proteomics ResearchGroup was supported by the Hungarian National Office for Researchand Technology (RET-08/2004). A participant of this consortium isKromat Ltd. that provided the Agilent 1100 nano-LC XCT Plus IonTrap mass spectrometer system. Special thanks to Christoph W. Turck(Max-Planck Institute of Psychiatry, Munich, Germany) who gave theopportunity to use the LTQ-Orbitrap Mass spectrometer in hislaboratory.

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