Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary
* Author for correspondence (e-mail: udvardy{at}nucleus.szbk.u-szeged.hu)
Accepted 18 December 2002
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Summary |
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Key words: 26S proteasome, Regulatory complex, S5a/Rpn10/p54 subunit, Multiubiquitin binding subunit, Mitotic phenotype
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Introduction |
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The functions of the RC can be deduced from a comparison of the enzymatic
properties of the 26S proteasome with those of the catalytic core. Protein
unfolding is probably one of the most important functions of RCs. The
chaperone-like activity of the RC may be responsible for protein unfolding
(Braun et al., 1999;
Strickland et al., 2000
).
Unfolding of the substrate proteins is most probably an ATP-dependent step,
and the six ATPase subunits present in the RC
(Dubiel et al., 1992
;
Dubiel et al., 1995
) may
perform the ATP hydrolysis required in this process. Opening of the central
channel of the catalytic core is performed by one of the ATPase subunits of
the RC, suggesting that channel opening is also an energy-dependent function
(Köhler et al., 2001
).
Although no direct experimental evidence is available, it is reasonable to
suppose that the feeding of unfolded proteins into the gated central channel
of the 20S proteasome is also an energy-dependent function of the RC. As the
20S proteasome is a non-specific protease, the selectivity of the 26S
proteasome towards multiubiquitinated proteins must be ensured by the RC. This
assumption is supported by the observation that S5a/Rpn10/p54 [for the
nomenclature of the human, yeast and Drosophila regulatory complex
subunits, see Hölzl et al.
(Hölzl et al., 2000
)] is
one of the RC subunits of the 26S proteasome that can recognise and bind
multiubiquitin chains in vitro (Deveraux et
al., 1994
; Deveraux et al.,
1995
; Haracska and Udvardy,
1995
; Haracska and Udvardy,
1997
; van Nocker et al.,
1996a
; van Nocker et al.,
1996b
). More recently, in vitro crosslinking studies revealed that
a reactive multiubiquitin chain can be selectively crosslinked to one of the
ATPase subunits of the RC (Lam et al.,
2002
). The role of S5a/Rpn10/p54 in substrate recognition is
debated owing to the observation that deletion of this subunit in yeast is not
lethal and has only a mild phenotype (Van
Nocker et al., 1996b
). Deletion of this subunit in the haploid
moss Physcomitrella patens, however, causes developmental arrest
(Girod et al., 1999
), and the
polyubiquitin-binding site of the fission yeast homologue of S5a/Rpn10/p54 is
essential when the S14/Rpn 12/p30 subunit is compromised
(Wilkinson et al., 2000
).
In order to gain an insight into the function of this RC subunit in higher eukaryotes, we generated a Drosophila mutant by deleting the single copy gene of subunit p54 (this gene is annotated in GadFly as pros54) and analysed the molecular changes and phenotypic effects of the deletion.
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Materials and Methods |
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Isolation of deletions by P-element-induced male
recombination
The P-lacW insertion near the 3' end of the pros54
gene in line 0554/18 (Deák et al.,
1997) was used to generate a mutant for the pros54 gene
by the male recombination system described by Preston et al.
(Preston et al., 1996
).
Egfr/CyO, P(
2-3) males were crossed en masse to
Gl Sb/TM3, Ser females. From the offspring the +/CyO,
P(
2-3); +/Gl Sb males were collected and crossed en masse
to homozygous yw; P-lacW0554/18 females. In the F0
generation, the yw/Y; +/CyO, P(
2-3); Gl Sb/+
P-lacW0554/18 + `jumpstarter males' were collected and crossed
in groups of 4-5 to 8-10 homozygous females of w1118
genotype. In the F1 generation, the w1118/Y; +/+; Gl
P-lacW0554/18 +/+ and the w1118/Y;
+/+; + P-lacW0554/18 Sb/+ recombinants were selected as
single males and crossed to yw; TM3, Sb/TM6, Hu females. In the F2
generation, the yw/Y; Gl P-lacW0554/18 +/ TM3, Sb males
were crossed to yw/w1118; Gl P-lacW0554/18 +/ TM3,
Sb females, or the yw/Y; + P-lacW0554/18 Sb/ TM6, Hu
males were crossed to yw/w1118; + P-lacW0554/18 Sb/
TM6, Hu females in order to establish stocks.
P-element-mediated transformation
The genomic HindIII, PstI and SacI rescue
constructs (Fig. 1C) were
microinjected together with the wing-clipped helper P-element
(wc2-3) into w1118 syncytial
blastoderm stage embryos by using standard techniques, and the
P(w+) transformants of the second generation were balanced
in stocks. For the rescue experiments, we used second chromosomal insertions
to allow the Df(3L)pros54P(w+) deletion on the third
chromosome to become homozygous.
|
Lethal phase analysis
The mutant genotype lacking pros54 [yw; Pst
I/y+CyO;
Df(3L)pros54P(w+)/Df(3L)pros54P(w+), see later] is
a segregant of the stock yw; Pst I/y+CyO;
Df(3L)pros54P(w+)/TM6c,Tb Sb. Eggs were collected from this
stock for 12 hours, and the first instar larvae were collected and transferred
to fresh food 24 hours later. The number of L2 and L3 larvae and puparia were
determined in parallel samples after 2, 4 and 6-8 days, respectively, taking
into consideration the fact that the mutant larvae developed at a lower rate.
To test their developmental capacity, white puparia (both mutant and
wild-type) were collected daily and transferred to a wet chamber to prevent
desiccation. Ages of pupae are given in hours after white puparium formation
(APF) at 25°C.
Embryo lethality was determined by counting the hatched and unhatched eggs laid by +/yw; Pst I/+; Df(3L)pros54P(w+)/+ parents derived from crossing yw/Y; Pst I/y+CyO; Df(3L)pros54P(w+)/TM6c, Tb Sb males to Oregon R females. The same experiment was repeated with +/w1118; Df(3L)pros54P(w+)/+ and Oregon R parents.
Cytological characterisation
Brains of wandering third instar larvae were dissected in PBS and
transferred into a drop of 45% acetic acid for 30 seconds. The brains were
then stained in a drop of 3% aceto-orcein (dissolved in 45% acetic acid) for
3-5 minutes, and the excess stain was removed by transferring the brains into
a drop of 60% acetic acid for a few seconds. Finally, the brains were
transferred into a small drop of 3% aceto-orcein (dissolved in 60% acetic
acid) on a coverslip, which was then picked up by touching it with a clean
microscope slide. The slides were wrapped in tissue paper and squashed very
hard for 10-15 seconds. The edges of the coverslip were sealed with nail
polish and the preparations were observed using a phase-contrast microscope,
using 40x and 100x objectives.
Protein gel electrophoresis and immunoblotting
Total protein extracts for denaturing polyacrylamide gel electrophoresis
(PAGE) were prepared by disrupting embryos, larvae or pupae directly in SDS
sample buffer in a microhomogeniser. The viscosity of the lysate was decreased
by shearing the extract through a 27 gauge injection needle. For immunoblot
analysis, proteins were separated on SDS-polyacrylamide gels, transferred to
nitrocellulose membrane, reacted with different subunit-specific monoclonal or
polyclonal antibodies and visualised by an enhanced chemiluminescent
technique, using HRP-conjugated secondary antibodies and the Supersignal-HRP
chemiluminescent substrate (Pierce).
Subunit-specific monoclonal antibodies were raised in mice immunised with
the purified regulatory complex. Hybridoma cell lines were selected by
standard procedures (Shulman et al.,
1978). The subunit specificity of the monoclonal antibodies had
been characterised previously (Kurucz et
al., 2002
).
For native polyacrylamide gel electrophoresis of total pupal protein
extracts, pupae were homogenised in a solution containing 20 mM Tris.Cl pH
7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM ATP, 1 mM DTT and 0.25 M
sucrose. After the extract had been cleared in a microcentrifuge by
centrifugation for 10 minutes, at 20,100 g at 4°C, the 26S
proteasome was analysed on the single layer native polyacrylamide gel system
described previously (Glickman et al.,
1998a). For immunoblotting, the gels were soaked for 5 minutes at
room temperature in western blotting transfer buffer supplemented with 1% SDS
and transferred to a nitrocellulose membrane by a standard procedure
(Sambrook et al., 1989
). The
in-gel dissociation of the proteasome subunits by SDS treatment greatly
improved the transfer efficiency, permitting the immunodetection of the 26S
proteasome from a single pupa or larva. DNA manipulations (cloning,
sequencing, PCR analysis, etc.) were carried out by standard procedures
(Sambrook et al., 1989
).
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Results |
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To obtain a null allele of pros54, we isolated a series of
chromosomal deletions generated by P-element-induced male
recombination as described previously
(Preston et al., 1996). Males
carrying the
2-3 transposase source on the second chromosome
and the 0554/18 P(w+) insertion at 78E on the third
chromosome over two dominant selectable markers (Gl to the left at
70C, and Sb to the right at 89B) were crossed to
w1118 homozygous females. From the offspring, the Gl
P(w+) + and the + P(w+) Sb recombinants
were selected as single males and crossed to females carrying balancers for
the third chromosome to establish lines. After a cross was found to be
fertile, the male was separated for DNA preparation.
Genomic DNAs were then screened for deletions to the left of the
P-element by PCR analysis with primer E located 380 bp upstream of
the pros54 transcription start site and primer PIR located in the
P-element inverted repeat (Fig.
1A). This primer pair gives a PCR product of 2.7 kb on the DNA of
the original line 0554/18 (data not shown). Among 30 recombinant lines showing
the Gl P(w+) + phenotype, we found three that gave a PCR
product shorter than 2700 bp, suggesting a deletion toward the left side
of the P-element. In one of these recombinants, the PCR product was
600 bp in length, indicating a
2100 bp long deletion between the
primer pair. Sequencing of the PCR product revealed that the exact size of the
deletion was 2095 bp (Fig. 1B),
and the deletion eliminated the whole of the coding region of the annotated
gene CG7181 and the 5' regulatory region and the first exon of
gene Vha M9.7-2 (CG7625), together with more than 90% of the coding
region of pros54. This means that in the deletion line, the 5'
end of pros54, including the regulatory region, the first exon and
intron and a short segment of the second exon, were retained; altogether,
these code for the first 29 amino acids of the p54 protein. Western blot
analysis (see later) revealed that the homozygous deletion
Df(3L)pros54P(w+) did not produce any detectable p54
protein and therefore could be considered to be a null allele.
To remove Gl and any possible background mutation by recombination, we crossed the Df(3L)pros54P(w+) line to w1118 flies, and the `purified' Df(3L)pros54P(w+) chromosome was balanced over TM3, Sb on a w1118 background. Deletion homozygotes of this line displayed lethality during the first and second larval stages.
Rescuing the functions of genes CG7181 and Vha
M9.7-2 (CG7625) removed by the Df(3L)pros54P(w+)
deficiency
In order to examine the real phenotype of the null allele of
pros54 alone, the other two genes affected by the deletion
Df(3L)pros54P(w+) should be rescued.
First, an 8 kb HindIII fragment derived from a 15 kb genomic clone
(Haracska and Udvardy, 1995)
that overlapped all three genes (Fig.
1C) was cloned into the transforming vector pP(CaSpeR-4)
and used for transformation (HindIII rescue construct). The flies
that were homozygous for Df(3L)pros54P(w+) and carried one
copy of the HindIII rescue construct were fully viable and fertile.
This proved that (i) the HindIII rescue construct is able to rescue
all three genes, and (ii) the deficiency chromosome has no other background
mutation.
Next, a 2.8 kb long genomic SacI fragment overlapping the pros54 gene (Fig. 1C) was cloned into the PstI site of the pP(W8) transforming vector (SacI rescue construct). The animals that were homozygous for the Df(3L)pros54P(w+) deficiency and carried one copy of the SacI rescue construct were early larval lethals, and none of them developed beyond the second larval stage. This means that the SacI rescue construct did not contain all the genetic information necessary for rescuing the Df(3L)pros54P(w+) deficiency.
Finally a 2.7 kb long PstI fragment of the same genomic clone overlapping the entire CG7181 and Vha M9.7-2 (CG7625) genes and a short 3' segment of pros54 (Fig. 1C) was also inserted into pP(CaSpeR-4). For injection into Drosophila w1118 embryos, we used a construct (named hereafter the PstI rescue construct) in which the region coding for the C-terminal 116 amino acids of p54 has an orientation opposite to that of the mini-w+ marker gene. Because of the lack of appropriate transcription and translation regulatory sequences, this construct cannot support the production of a truncated C-terminal p54 protein product. As all these constructs were made of genomic fragments, they contained the authentic regulatory sequences allowing correct spatial and temporal expression of the genes.
Homozygous flies for the Df(3L)pros54P(w+) deficiency
that carried one copy of the PstI rescue construct and one copy of
the SacI rescue construct were fully viable and fertile. This showed
that the PstI and the SacI rescue constructs together
contained all the genetic information removed by the
Df(3L)pros54P(w+) deficiency. When the PstI
rescue construct was in combination with the homozygous
Df(3L)pros54P(w+), the animals exhibited larval-pupal
lethality (see later). This combination [yw; Pst/y+CyO;
Df(3L)pros54P(w+)/Df(3L)pros54P(w+)] lacked
pros54 but had full copies of the CG7181 and Vha
M9.7-2 (CG7625) genes. Therefore, the larval-pupal lethality was a
consequence of the deletion of pros54 alone and represents the
p54-null phenotype. From here on, this combination will be denoted
p54.
Deletion of pros54 results in larval-pupal polyphasic
lethality
p54 mutant animals display polyphasic lethality during
their development. In the embryonic phase, mortality is apparently similar to
that in the control, that is, most of the embryos hatched as first instar (L1)
larvae (data not shown). During larval development, there is an increase in
mortality (Table 1). The
surviving larvae develop more slowly than the wildtype, and the majority of
them reach maturity and pupariate 3-4 days later than their heterozygous
siblings. Although the size of the mutant larvae is almost normal, some of
their internal organs are significantly smaller, especially in the
late-pupariating ones. For example the brain and the ventral ganglion are
about half of the normal size (Fig.
2G). Only 27% of the hatched larvae survive up to the end of
larval development and form puparia. These mutant puparia are smaller than the
wild-type ones and have a characteristic bent shape
(Fig. 2A,B). The cuticle of the
mutant puparia is softer, lighter in colour and not so rigid as the wild-type
one, suggesting incomplete tanning of the mutant cuticle. In about one-third
of the puparia, a complete pupal cuticle is secreted, which fully covers the
head, thorax and abdomen. However, the head eversion is mostly incomplete and
the appendages (wings and legs) are always smaller and shorter than normal
(Fig. 2D).
p54
mutant pupae never developed beyond this stage. In half of the puparia, only
imaginal disc derivatives (head, thorax, adult appendages and the region of
the external genitalia) secrete the pupal cuticle. In the later examples, the
pupal cuticle is either missing or incomplete on the abdomen
(Fig. 2B). In extreme cases
(10% of the specimens), some pupal cuticles can only be found in the regions
of the head and the external genitalia
(Fig. 2F). All the mutant
puparia dry out in 1-2 days, suggesting that the puparial cuticle can not
prevent desiccation, unlike the situation in the wildtype. We successfully
prevented desiccation by keeping the mutant puparia in a wet environment. Even
if they remained alive for a longer time, they could not develop further and
they finally died. For example, the mutant specimens in
Fig. 2A,B,D,F were kept in a
wet chamber for 60 hours before the pictures were taken.
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|
Histolysis of the larval tissues (salivary gland, midgut epithelium, body wall muscles, etc.) apparently occurs, and the fat body breaks up into single cells (Fig. 2B,F). The metamorphosis seems to stop at this point because the inner organs of the adult are not formed and the secretion of an adult cuticle with hairs and bristles never takes place. It should be noted that limited cell proliferation could sometimes be observed in the imaginal rudiments of the midgut epithelium (imaginal islands and imaginal ring at the midgut-hindgut boundary). These tissues contain small cells with small diploid nuclei that could easily be distinguished from the large polythenic larval cells by DAPI staining (data not shown).
Deletion of pros54 results in multiple mitotic defects
The activity of the proteasome is essential for normal cell cycle
progression. To determine the role or contribution of subunit p54 to the
overall function of the proteasome in the cell cycle, we analysed neuroblast
preparations from larvae lacking this subunit.
The examination of mitotic cells in squashed preparations of the central
nervous system from p54 third instar larvae revealed several
characteristic features. First, the mitotic index in
p54
preparations is increased compared with that in the wildtype
(Table 2). The frequency of
prometaphase and metaphase forms is also higher in the mutant. Additionally, a
significant proportion of
p54 mitotic cells have
over-condensed chromosomes (Fig.
3B), similar to those caused by colchicine treatment. These
features arose as a consequence of mitotic arrest and indicate that
p54 cells can enter mitosis, but their progression through and
exit from mitosis is delayed or blocked for some time. Moreover, a significant
proportion of the cells in prometaphase and metaphase show no obvious
centromeric connection between at least some of the sister chromatides
(Fig. 3F), which indicates
premature sister chromatid separation. Some of the cells in anaphase display
chromosome bridges and lagging chromosomes. Characteristically, in about 19%
of mitotic cells all major chromosomes are arranged in a circle with the
centromeres pointing toward the centre, and the dot-like fourth chromosomes
are always located in the middle of the circle
(Fig. 3C). These circular
mitotic figures (CMFs) are similar to CMFs found in mgr, polo and
aur mutants in Drosophila
(Gonzalez et al., 1988
;
Sunkel and Glover, 1988
;
Glover et al., 1995
), where it
was suggested that they are caused by monopolar spindles. Monopolar spindles
are formed as a consequence of failure(s) in centrosome duplication and/or
separation. A further characteristic feature of
p54 mutants is
the high frequency of aneuploid (Fig.
3B,E) and polyploid (Fig.
3D) cells. The existence of tetra- and octaploid cells suggests
that they were able to escape mitotic arrest and undergo further cell cycle(s)
without chromosome segregation or cytokinesis. The frequency of all these
abnormal mitotic figures is summarized in
Table 2.
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|
Molecular analysis of the 26S proteasome present in the
p54 pupae
An immunoblot assay with subunit-specific monoclonal antibodies
unequivocally proves the complete loss of subunit p54 in p54
pupae. In a total protein extract prepared from a single 24-hour-old wild-type
pupa, all four subunits, including p54, can be detected in an immunoblot assay
by using four different subunit-specific monoclonal antibodies
(Fig. 4, lane 1). This is in
sharp contrast to the immunoblot pattern obtained on the
p54
pupal protein extract (lane 2), in which the p54 subunit was not detected. The
absence of p54 protein in the deletion mutant is in agreement with the results
of PCR amplification experiments performed with different primer pairs and the
genomic DNA of strains carrying different rescue constructs (data not
shown).
|
We have demonstrated previously that the 26S proteasome is present at a
very high concentration in Drosophila embryos, and its concentration
declines during the larval stages of development
(Udvardy, 1993). The high
concentration of the 26S proteasome in 0- to 2-hour-old embryos indicates that
its deposition is due to a maternal effect, and the maternally stored
proteasome particles are gradually depleted during the larval developmental
stages. The developmental profile of the 26S proteasome during the pupal
stage, however, has not been tested previously. The abundance of the 26S
proteasome was followed by an immunoblot assay during the
embryonic-larval-pupal developmental stages of the Oregon R wild-type
Drosophila strain. Protein extracts were prepared from a single third
instar larva, a single pupa of different ages and 0- to 12-hour-old embryos.
The protein extracts were fractionated by SDS-PAGE (9% gel) and immunoblotted
with a mixture of two monoclonal antibodies specific for subunits p54 and
p48A. The embryonic extract was prepared from 1 mg of embryo, which is the
average weight of a third instar larva or a pupa. As shown in
Fig. 5, the concentration of
the 26S proteasome is very high in the embryos; it is very low in the third
instar larvae (after a short exposure, it is not detected in a single third
instar larva, but it can be detected after a longer exposure; data not shown),
and its concentration increases sharply during the first 4 hours of pupal
development. This sudden increase in the 26S proteasome concentration may be
essential to support the sharp increase in mitotic activities of imaginal
discs during the larval-pupal developmental transition. The increased demands
of the proteasomal activity and the compromised function of the mutant 26S
proteasome may be the reasons for the observed lethality.
|
Immunoblot analysis with an anti-ubiquitin antibody revealed that there is no significant increase in the total amount of multiubiquitinated proteins in the deletion mutant. However, there was a shift in the proportion of highly multiubiquitinated proteins in the pupae of the deletion mutant (Fig. 6). This shift may be a consequence of either an upregulation of the enzyme cascade responsible for the multiubiquitination of proteins or a downregulation of the deubiquitinating enzyme activity in the mutant cells. It is more probable, however, that the accumulation of highly multiubiquitinated proteins is the manifestation of an impaired degradation of a certain class of multiubiquitinated substrate proteins.
|
In Saccharomyces cerevisiae, deletion of the S5a/Rpn10/p54 subunit
destabilises the RC of the 26S proteasome, which comes apart into lid and base
subcomplexes, during the purification procedure
(Glickman et al., 1998b). To
test the stability of the mutant Drosophila RC, freshly prepared
protein extracts from 4-to 24-hour-old wild-type or mutant pupae were
fractionated on a native polyacrylamide gel, and the integrity of the 26S
proteasome was analysed by an immunoblot technique. It is known that the
intact 26S proteasomes can be resolved into two distinct isoforms by native
PAGE. These isoforms probably correspond to the singly capped and the doubly
capped forms of the enzyme seen in the electron microscope
(Glickman et al., 1998a
;
Hölzl et al., 2000
). The
electrophoretic pattern of the mutant 26S proteasome was indistinguishable
from that of the wild-type enzyme (Fig.
7). Both in the wild-type and in the mutant pupal extracts,
monoclonal antibodies specific for subunits of either the lid or the base
subcomplexes stained both isoforms with equal intensity, indicating that in
these bands complete RCs and not lid or base subcomplexes are present.
Furthermore, the bands that are recognised by the lid- and the
base-subcomplex-specific monoclonal antibodies also reacted with a polyclonal
antibody specific for the catalytic core, indicating that both bands
correspond to the 26S proteasome. Thus, deletion of subunit p54 does not
destabilize the RC and does not interfere with the assembly of the RC and the
catalytic core in Drosophila. The electrophoretic mobilities of the
26S proteasome isoforms obtained from wild-type or
p54 strains
are very similar. Because of the high resolving power of native PAGE, this
indicates that the absence of subunit p54 does not induce a structural
rearrangement in the 26S proteasome extensive enough to influence the
electrophoretic mobility of the particle. Free RC [running between the
26SII and the 20S proteasome
(Hölzl et al., 2000
)] is
not present at a detectable level in the mutant pupae. Although native PAGE is
only an analytical method, with obvious limitations in the sensitivity of
detection, the lack of immunoreactive material in the lower part of the gel
indicates that neither free subunits nor partially assembled particles are
present in significant quantity in mutant pupae.
|
The increased demands of the proteasomal activity during the pupal
developmental stage, and the compromised function of the mutant 26S proteasome
in p54, allowed the study of a specific aspect of the
regulation of expression of the genes encoding proteasomal subunits. Assuming
a feedback regulatory circuit, in which increased proteasomal activity demands
induce the upregulation of the expression of genes coding for proteasomal
subunits, one would expect a higher 26S proteasome content in the mutants, or
at least the upregulation of those subunits involved in the coordinated
feedback regulation. To compare the 26S proteasome contents of the wild-type
and
p54 pupae, single 24-hour-old pupae from both strains were
disrupted directly in SDS sample buffer and analysed by the immunoblot
technique with polyclonal antibodies raised against either the highly purified
RC or the highly purified 20S proteasome. As shown in
Fig. 8, there are huge
differences in both the RC and the 20S proteasome content of the wild-type and
p54 pupae. Densitometric analysis revealed that the RC
contents of the
p54 pupae is at least 20-fold higher than that
of the wildtype. This difference is not due to an unequal loading of the
proteins, because no difference in immunoblot intensities was found when
antibodies specific for two different household proteins were used on the same
filter (Fig. 8, lanes 5-8). It
was even more surprising that both the RC and the catalytic core of the 26S
proteasome exhibited an extreme upregulation in the deletion mutants
(Fig. 8, lanes 1-4).
|
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Discussion |
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The discovery that the DNA repair protein Rad 23 carries an N-terminal
ubiquitin-like domain (UBL) that interacts with the 26S proteasome initiated
an alternative approach to the understanding of the selective substrate
recognition mechanism of the 26S proteasome
(Watkins et al., 1993;
Schauber et al., 1998
;
Hiyama et al., 1999
). Besides
the UBL domain, Rad 23 contains two partially homologous sequence motifs, the
ubiquitin-associated domains (UBA), present in several cellular proteins,
which can recognise and bind ubiquitin moieties
(Hofmann and Bucher, 1996
;
van der Spek et al., 1996
;
Chen et al., 2001
). Originally,
monoubiquitin was considered to be the interacting partner of the UBA domain
(Bertolaet et al., 2001
), but
more recent data indicate that the multiubiquitin chains are bound
preferentially (Wilkinson et al.,
2001
; Rao and Sastry,
2002
). It is believed that proteins carrying both UBL and UBA
domains are involved in the substrate selection for the proteasome. The UBA
domain is required for the selective recognition and binding of
multiubiquitinated proteins, whereas the UBL domain generates the interaction
with the 26S proteasome, a prerequisite for presenting the multiubiquitinated
substrate proteins for degradation. This assumption is supported by the
observation that not only Rhp 23 (the fission-yeast homologue of Rad 23) but
also another fission yeast protein, Dph1, which carries both UBA and UBL
domains, has the same dual properties: it can specifically recognise and bind
the multiubiquitin chains and interact with the 26S proteasome
(Wilkinson et al., 2001
). The
UBL domain of Rad 23 interacts with the S5a/Rpn10/p54 subunit of the RC
(Hiyama et al., 1999
). The
coordinated role of the UBA-UBL-containing proteins and the S5a/Rpn10/p54
subunit in substrate selection is supported by the observation that the single
and double deletion mutants of Rhp 23, Dhp1 and Pus 1 (the fission-yeast
orthologue of S5a/Rpn10/p54) are viable, whereas triple deletion of these
genes was lethal (Wilkinson et al.,
2001
). Thus, UBA-UBL-containing proteins in co-operation with
S5a/Rpn10/p54 are indispensable for the degradation of essential proteins, and
the stabilisation of these proteins is lethal for the cell.
Nevertheless, the role of S5a/Rpn10/p54 in substrate selection is still controversial. If its ubiquitin-binding function is required only for the targeting of UBL-containing proteins to the proteasome, why does it show strict preference for multiubiquitin chains in an in vitro ubiquitin-binding assay, although there is only one single ubiquitin moiety in the UBL domain? Furthermore, if it is assumed that UBA-containing proteins are the true multiubiquitin chain receptors, and the only role of S5a/Rpn10/p54 in substrate selection is its ability to interact with the UBL domain of UBA-containing proteins, why is the yeast S5a/Rpn10/p54 deletion mutant viable? It is more reasonable to suppose that there are structurally distinct classes of multiubiquitinated proteins, which are recognised and targeted for degradation by distinct but partially overlapping mechanisms. Certain multiubiquitinated proteins may be selected and targeted exclusively by the Rpn10/S5a subunit. For the recognition of other classes of proteasome substrates, UBA-containing receptors are required. Targeting of these substrates may require an UBL domain in the receptor, which alone or in cooperation with unmasked ubiquitin moieties of the multiubiquitin chain may interact with the S5a/Rpn10/p54 subunit, promoting the targeting of the substrate to the proteasome.
To explain the viable phenotype of the yeast S5a/Rpn10/p54 deletion mutant,
alternative substrate recognition and targeting mechanisms must be considered.
Database analyses have identified eight proteins with a UBA domain in the
fission yeast genome (Wilkinson et al.,
2001). All eight UBA proteins are able to bind multiubiquitin
chains, but only two of them contain an additional UBL domain. The
interactions of UBA proteins (without an additional UBL domain) with the 26S
proteasome have never been tested. It may be assumed that these UBA proteins
may target multiubiquitinated proteins to the proteasome by interacting with
RC subunits other than the S5a/Rpn10/p54; this is a plausible alternative,
which may explain the viable phenotype of the yeast S5a/Rpn10/p54 deletion
mutant. This assumption is supported by the observation that, although Pus 1
is not required for cell viability in the fission yeast, deletion of Pus 1 is
synthetically lethal with mutations of three other RC subunits (Rpn12, Rpn11
and Rpn1). Overexpression of the wild-type Pus 1 protein, but not its mutant
version without multiubiquitin-binding activity, could rescue a
temperature-sensitive mutation of Rpn12
(Wilkinson et al., 2000
).
Moreover, the close physical association of Pus 1 and Rpn12 proteins has been
demonstrated, suggesting their cooperation in substrate selection.
The hypothesis of a direct and unaided role of S5a/Rpn10/p54 as a
multiubiquitin receptor in yeast is supported by the observation that the
degradation of certain proteasome substrates is impaired in the yeast
Df(S5a/Rpn10/p54) mutant (van Nocker et
al., 1996b). This observation supports the notion that
S5a/Rpn10/p54 functions as a multiubiquitin receptor for certain substrate
proteins, and no other protein is involved in this function. The mild
phenotype of this mutant, however, suggests that the number of
multiubiquitinated proteins recognised and targeted exclusively by this RC
subunit in the yeast is limited. The lethality of
p54
indicates that in Drosophila either the number of multiubiquitinated
proteins processed exclusively by the S5a/Rpn10/p54 subunit is much larger or,
during the pupal developmental phase, a few key substrate proteins have to be
processed exclusively by this RC subunit, and insufficient degradation of
these proteins can block the developmental program, resulting in lethality.
The severe mitotic defects observed in the larval brain of the mutants suggest
that proteins involved in the cell cycle regulation may belong to this
specific class of substrate proteins.
The viability of p54 embryos and larvae is due to a large
pool of maternally stored 26S proteasomes in the embryos, which becomes only
gradually depleted during the larval stage. Thus, it cannot be stated that the
S5a/Rpn10/p54 subunit is essential for the appropriate functioning of the
proteasome in every cell, through all phases of the development, or that,
similarly to the yeast cells, it is generally indispensable but essential only
in certain phases of the development. The polyphasic larval-pupal lethality of
the present mutant, however, suggests that, as soon as the maternally stored
wild-type 26S proteasome depot is depleted, mutant proteasomes, even in
excess, can not rescue the lethality.
Recently it has been shown that in mice, Rpn10 mRNA is present in at least
five distinct developmentally regulated alternatively spliced forms. Protein
products of these forms are components of the 26S proteasome, with an
apparently similar affinity for multiubiquitinated lysozyme
(Kawahara et al., 2000).
RT-PCR analysis of polyA+ RNAs prepared from Drosophila
embryos, pupae and flies revealed a single mRNA product (data not shown). Thus
the pupal lethality of our mutant is not a consequence of the elimination of a
pupal-specific form of the p54 mRNA, which can not be complemented with other
spliced variants of the p54 mRNA.
The undisturbed assembly of the RC and the catalytic core, and the lack of
gross structural disintegration of the 26S proteasome in the
p54 animals strongly suggest that the pupal lethality of the
mutant is due to the impairment of some specific function of the proteasome
owing to the lack of subunit p54.
The specific crosslinking of a reactive version of a tetraubiquitin chain
to the S6'/Rpt5/p50 ATPase subunit
(Lam et al., 2002) suggests
the involvement of this subunit in substrate selection. This observation,
however, does not exclude a similar role for other subunits in this process.
The crosslinking of two polypeptides depends on the optimum spatial
configuration of two reactive side-chains of the interacting polypeptides,
which are specific for the applied crosslinker. If the distance between these
reactive side-chains is out of the range of the spacer arm of the crosslinker,
covalent crosslinking cannot occur, even between strongly interacting
polypeptides. This question will ultimately be settled by the identification
of cellular proteins processed selectively by the different recognition
mechanisms.
In the yeast, RPN4 was identified as a transcription factor involved in the
coordinated regulation of genes encoding proteasomal subunits
(Mannhaupt et al., 1999;
Xie and Varshavsky, 2001
).
RPN4 is a very short-lived protein, a substrate of the 26S proteasome, which
interacts with the RC subunit Rpn2. The observations that RPN4 can
coordinately enhance the expression of proteasomal genes, and that at the same
time it is degraded by the proteasome, led to the supposition of a feedback
circuit (Xie and Varshavsky,
2001
). In this circuit, RPN4 upregulates the expression of genes
encoding proteasome subunits, and it is finally destroyed by the assembled
active proteasomes. In higher eukaryotes, the coordinated regulation of genes
encoding proteasomal subunits has never been demonstrated. If a similar
feedback circuit operates in higher eukaryotes, the accumulation of
proteasomal subunits would be expected under conditions when the demand for
proteasomal activity increases and/or the presumed regulator of the circuit is
stabilised. The extreme accumulation of proteasomal subunits in the
p54 animals lends strong support to the existence of such a
feedback circuit. The large mass of the proteasomal subunits in this mutant is
present in the form of fully assembled proteasomal particles. The lack of free
subunits and/or partially assembled proteasomal complexes is a direct
indication of a fully coordinated regulation of the expression of all
proteasomal subunits. A transcription factor homologous to the yeast RPN4 has
not hitherto been identified in higher eukaryotes. The extreme and coordinated
overexpression of the proteasomal subunits in
p54 mutant,
however, indicates that a transcription factor(s) capable of coordinately
regulating the expression of proteasomal genes must also function in higher
eukaryotes.
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