1. Occurrence of a Putative SCF Ubiquitin
Ligase Complex in Drosophila
Silvia N. Bocca,*,1
Mariana Muzzopappa,*,1
Susana Silberstein,† and Pablo Wappner*,2
*Instituto de Investigaciones Bioquı´micas “Fundacio´n Campomar,” Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Patricias Argentinas 435, 1405, Buenos Aires, Argentina; and †Instituto de
Investigaciones Biotecnolo´gicas, Universidad de San Martin, San Martin, Pcia. de Buenos Aires, Argentina
Received July 10, 2001
Many proteins are targeted to proteasome degrada-
tion by a family of E3 ubiquitin ligases, termed SCF
complexes, that link substrate proteins to an E2
ubiquitin-conjugating enzyme. SCFs are composed of
three core proteins-Skp1, Cdc53/Cull, Rbx1/Hrt1-and a
substrate specific F-box protein. We have identified in
Drosophila melanogaster the closest homologues to
the human components of the SCF␤TrCP
complex and
the E2 ubiquitin-conjugating enzyme UbcH5. We show
that putative Drosophila SCF core subunits dSkpA
and dRbx1 both interact directly with dCu11 and the
F-box protein Slmb. We also describe the direct inter-
action of the UbcH5 related protein UbcD1 with dCul1
and Slmb. In addition, a functional complementation
test performed on a Saccharomyces cerevisiae Hrt1p-
deficient mutant showed that Drosophila Rbx1 is able
to restore the yeast cells viability. Our results suggest
that dRbx1, dSkpA, dCullin1, and Slimb proteins are
components of a Drosophila SCF complex that func-
tions in combination with the ubiquitin conjugating
enzyme UbcD1. © 2001 Academic Press
Key Words: SCF; ubiquitin; Drosophila; proteolysis;
E3 ligase.
The ubiquitin/proteasome pathway plays a key role
in many basic cellular processes (reviewed in 1). In
addition to degradation of defective or misfolded pro-
teins, a critical regulatory role for this system has been
defined in studies of the cell cycle. Some major signal
transduction pathways, which are of great importance
in development and cell differentiation, are known to
be controlled in the same way. How do eukaryotic cells
decide which proteins are due to enter the ubiquitin/
proteasome pathway and when such proteins should do
so? Typically, the conjugation reaction occurs in three
successive steps. First, Ubiquitin Activating Enzyme
(E1) hydrolyzes ATP to form an AMP-ubiquitin adenyl-
ate. After release of AMP, activated ubiquitin is trans-
ferred to the active site cysteine of E1 to form an
E1-thiolester bond. Second, activated ubiquitin is
transferred from E1 to an Ubiquitin Conjugating En-
zyme (E2) in a reaction in which the thiolester-bond is
transferred. Finally, a Protein Ubiquitin Ligase (E3)
transfers ubiquitin from E2 to a lysine group on the
target protein. A multi-ubiquitin chain is generated,
targeting the protein for degradation by the 26S pro-
teasome.
Results from several laboratories have demonstrated
that ubiquitin conjugation is the step that defines tar-
get specificity for degradation, and that E3 protein-
ubiquitin ligases are the enzymes responsible for such
specificity. Although some E3 enzymes are single
polypeptides, multiprotein complexes appear to be
most common. Among these complexes, SCF E3 ubiq-
uitin ligases have been well characterized in mamma-
lian and yeast systems (2). They are composed of three
constant polypeptides—Skp1, Cdc53/Cullin1 (Cul1)
and Rbx1/Hrt1—and one variable component (F-box
protein), which is specific to a particular target or small
group of target proteins. Evidence provided by several
laboratories indicate that F-box proteins behave as
adapters between substrates and the rest of the com-
plex (3). The so-called “Combinatorial F-box Hypothe-
sis” proposes that many different substrates can be
recognized by various SCF complexes that differ in
their F-box adapter protein (3–5). A mammalian SCF
E3 ubiquitin ligase complex that contains an F-box
protein named ␤TrCP controls NF␬B and Wnt/
Wingless signaling pathways (6). Such control is ex-
erted through ubiquitination of I␬B and ␤-catenin, re-
spectively thus, targeting them for 26S proteasome
degradation. Although the existence of SCF complexes
has not been demonstrated in Drosophila, database
sequences analysis indicates that many F-box proteins
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed at Instituto de
Investigaciones Bioquı´micas “Fundacio´n Campomar,” Patricias Ar-
gentinas 435, 1405, Buenos Aires, Argentina. Fax: (54 11) 4865
2246/4863 1916. E-mail: pwappner@leloir.org.ar.
Biochemical and Biophysical Research Communications 286, 357–364 (2001)
doi:10.1006/bbrc.2001.5394, available online at http://www.idealibrary.com on
357 0006-291X/01 $35.00
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.

2. occur in the fruit fly. Slimb (Slmb), the Drosophila
homologue of ␤TrCP is a negative regulator of Wing-
less and Hedgehog signaling pathways during limb
development (7, 8) and also seems to control NF␬B
pathway (9). Slmb loss of function mutations result in
a cell-autonomous accumulation of high levels of Cub-
itus interruptus (Ci), a transcription factor that acti-
vates the expression of Hedgehog responsive genes and
Armadillo (Arm), the Drosophila ␤-catenin homologue
(8). These results suggest that Slmb plays an analo-
gous role to mammalian ␤TrCP, targeting Arm, Ci, and
Cactus (the I␬B Drosophila homologue) for ubiquitina-
tion and proteolysis by the 26S proteasome. In addi-
tion, Slmb was also shown to be required for proper
centrosome duplication in Drosophila causing the ap-
pearance of additional centrosomes and mitotic defects
in mutant larval neuroblasts (10).
Recently, a cul1/cdc53 Drosophila homologue gene
has been described (11) and several sequences homol-
ogous to vertebrate and yeast skp1 and rbx1/hrt1 are
present in the Berkeley Drosophila Genome Project
database. Nevertheless, no biochemical evidences for
the occurrence of SCF complexes in Drosophila were
presented so far. In this report, we provide evidence for
the existence of a Slmb-containing SCF complex in the
fruit fly by utilizing the yeast two-hybrid system as
well as in vitro interactions. In addition, we tested the
ability of skpA, cul1, and rbx1 Drosophila genes
(dskpA, dcul1, and drbx1) to complement the corre-
sponding null mutations in Saccharomyces cerevisiae.
We found that drbx1, but not the other two components
of the Drosophila SCF complex, functionally comple-
ments ⌬hrt1, thus indicating that Rbx1 is largely con-
served in evolution.
MATERIALS AND METHODS
DNA database searches. Drosophila sequences with the highest
identity to the human proteins Skp1, Rbx1, and Cul3 (GenBank
Accession Nos: U33760, AF140598, and AF062537, respectively)
were identified at the Berkeley Drosophila Genome Project (BDGP)
database using BLAST and designated as dSkpA, dRbx1, and dCul3.
The identification of dCul1 was recently reported (11). To obtain the
corresponding cDNAs, the BDGP Drosophila EST database was
searched and several Drosophila EST clones were found to encode
proteins matching the 5Ј end of the identified sequences. cDNA
clones corresponding to the Drosophila F-box protein Slimb
(AF032878), dCul1 (Q24311), and the Drosophila E2 ubiquitin-
conjugating enzymes UbcD1 (12) (X62575), UbcD6 (13) (M63792),
and UbcD10 (14) (AJ238007) were obtained from the same source.
One representative EST clone was selected between those matching
the cDNA sequences corresponding to dSkpA (Clone ID: LP02056),
dRbx1 (Clone ID: GH12110), dCul1 (Clone ID: LD29973), dCul3
(Clone ID: GH27416), Slmb (Clone ID: LD31459), UbcD1 (Clone ID:
LD32571), UbcD6 (Clone ID: LD22830), and UbcD10 (Clone ID:
LD30207) and used for experiments. Clones were sequenced by au-
tomated DNA sequencing to confirm that all ESTs contained the
full-length coding region. The sequences of dSkpA, dRbx1, and dCul3
correspond to the Flybase annotations: gene CG16983, (transcript
CT40292), gene CG16982 (transcript CT32695), and gene CG11861
(transcript CT35206), respectively.
Plasmid construction. cDNAs were amplified by PCR using oli-
gonucleotides that introduced restriction enzyme sites for in-frame
cloning of the PCR products into pTrcHisC (Invitrogen) or pGEX-3X
plasmids (Pharmacia).
In vitro transcription and translation. [35
S] methionine-labeled
proteins were produced from ESTs cloned in the pOT2 vector by
coupled in vitro transcription and translation (IVTT) reactions in
rabbit reticulocyte lysate (TNT, Promega).
Expression of recombinant proteins in E. coli. Cultures of E. coli
DH5␣ bearing GST- or His-fusion protein encoding plasmids were
grown to an A600 of 0.5. Induction was performed by addition of IPTG
was added to a final concentration of 50–100 ␮M. Cultures were
allowed to grow for 2.5 h at 28°C, and cells were harvested by
centrifugation at 4°C.
Purification of GST-fusion proteins. Bacterial pellets were lysed
on ice by sonication in resuspension buffer (PBS, 1% Triton X-100,
0.5 mM EDTA) containing a protease inhibitor cocktail (0.1 mM
phenylmethylsulfonylfluoride, 1 ␮g/1 ml leupeptin, 1 ␮g/1 ml pep-
statin, and 1 ␮g/1 ml aprotinin). Lysates were centrifugated, super-
natants mixed with glutathione-agarose beads (Sigma) (200 ␮l beads
per 50 ml of initial bacterial culture) were allowed to bind for 1 h at
4°C. Glutathione-agarose beads were pelleted by low speed centrif-
ugation and washed five times in resuspension buffer. For binding
assays (below), the concentration of GST fusion proteins in the slurry
of glutathione-agarose beads was estimated by SDS–PAGE and Coo-
massie blue staining.
Glutathione-agarose pull-down assay. GST-fusion proteins bound
to glutathione-agarose (ϳ20 ␮l beads) were mixed with 2 ␮l of
[35
S] methionine-labeled protein generated by IVTT, and allowed to
interact at 4°C for 3 h in 100 ␮l of EBC buffer (50 mM Tris–HCl, pH
7.5, 150 mM NaCl, 0.5% v/v Igepal, 0.5 mM EDTA). Glutathione-
agarose beads were pelleted by low speed centrifugation and washed
five times in EBC buffer at 4°C. Samples were resuspended in
SDS-sample buffer and associated proteins were detected by SDS–
PAGE followed by fluorography.
Purification of His-fusion proteins. Bacterial pellets were lysed
on ice by sonication in binding buffer (20 mM Tris–HCl pH 7.9, 0.5 M
NaCl, 5 mM imidazole) with a protease inhibitors cocktail. Lysates
were centrifuged and the supernatants were mixed with iminodiace-
tic acid-sepharose beads (Sigma) previously charged with Ni2ϩ
. The
beads were washed with 10 volumes of binding buffer followed by 6
volumes of washing buffer (20 mM Tris–HCl pH 7.9, 0.5 M NaCl, 60
mM imidazole). Bound His-fusion proteins were recovered with 6
volumes of elution buffer (20 mM Tris–HCl pH 7.9, 0.5 M NaCl, 1 M
imidazole).
In vitro interaction assays using His-tagged proteins. His-tagged
purified proteins (100 ␮l) were mixed with 4 ␮l of rabbit reticulocyte
lysate containing [35
S] methionine-labeled protein and 400 ␮l of EBC
buffer and allowed to interact for 3 h at 4°C. Samples were incubated
for 2 h at 4°C with 200 ng of anti-His antibody (H-15) (Santa Cruz
Biotechnology) and immunoprecipitation was performed by addition
of 20 ␮l of protein A-Sepharose (Sigma) followed by low speed cen-
trifugation. After washing the beads five times in EBC buffer, sam-
ples were dissolved in sample buffer and associated proteins were
detected by SDS–PAGE followed by autoradiography.
Yeast two-hybrid interactions. pAS2-1 plasmid (Clontech) was
used to generate fusion proteins containing the GAL4 DNA-binding
domain (DNA-BD) at their N-terminal end and pACT2 vector (Clon-
tech) to generate proteins containing the GAL4 activation domain
(AD) with a hemagglutinin (HA) peptide at their N-terminal end.
Expression in yeast was confirmed by Western blot using anti-GAL4-
DBD (IKE) or anti HA-probe (Y-11) antibodies (Santa Cruz Biotech-
nology) (data not shown). The PJX yeast strain that contains three
reporter elements (HIS3, ADE2, and LacZ) was transformed with
various combinations of recombinant pACT2 and pAS2-1 plasmids
and plated on SD medium lacking leucine and triptophane to select
Vol. 286, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
358

3. for the presence of both plasmids. Interactions were considered pos-
itive when the three reporters were expressed. ␤-galactosidase ex-
pression was assayed by streaking on media containing X-Gal as well
as in liquid culture assays by the ONPG colorimetric method. ADE2
and HIS3 expression was assayed as growth on SD medium lacking
leucine, triptophane, adenine, and histidine (Clontech).
Yeast mutant strains and plasmids used in complementation
studies. Diploid strains CSLZ019(HE), CD53/cdc53; relevant geno-
type: CEN.PK2, YDL132W/ydl132w (1,2447)::KanMX4, and CEN.PK2
were obtained from EUROSCARF (Frankfurt).
Construction of ⌬skp1 and ⌬rbx1 yeast strains. A PCR-based
gene disruption method (15) was used to produce linear fragments
containing a heterologous HIS3 fragment flanked by 5Ј and 3Ј un-
translated homology regions from the SKP1 and HRT1 genes. The
pFA6a-HIS3MX6 plasmid used for PCR-based gene disruptions was
provided by Dr. Peter Philippsen (University of Basel, Switzer-
land). Diploid strain CEN.PK2 was transformed to histidine proto-
trophy using the PCR products to generate strains SWY100
(SKP1/⌬skp1::HIS3), and SWY200 (HRT1/⌬hrt1). Diploid strain
YPH274 (ATCC, Rockville, MD) was also transformed with PCR
fragments to generate heterozygous diploids with null alleles of
SKP1 and HRT1. Correct allele replacement with HIS3 was con-
firmed by diagnostic PCR in all the strains (15).
Functional complementation of yeast strains. Full-length dCul1,
dSkpA, and dRbx1 cDNAs were subcloned into multicopy plasmids
URA3-marked p426, under control of the constitutive promoter GPD
(16) and pYES2, under control of the glucose-repressible galactose-
inducible GAL1-10 promoter (Invitrogen, San Diego, CA). Yeast
strains were electroporated and uraϩ
transformants were selected on
synthetic media lacking uracil. Tetrads from sporulated cultures
were dissected on plates containing 2% glucose or 4% galactose as a
carbon source, and incubated at 24°C for 2–4 days. The expression of
dCul1 and dSkp1 in yeast was also performed with constructs bear-
ing the Drosophila cDNAs under control of two high-expression
(constitutive or regulatable) promoters and tested for complementa-
tion of strains CSLZ019(HE) and SW100, respectively. Complemen-
tation with Drosophila dSkp1 was also tested on SKP1/⌬skp1::HIS3
constructed on strain YPH274.
RESULTS AND DISCUSSION
To identify the Drosophila components of a putative
SCF complex we searched the Berkeley Drosophila
Genome Project (BDGP) database with the human
Skp1 and Rbx1 protein sequences. BLAST search us-
ing hSkp1 yielded 7 Drosophila Skp1-related proteins.
Like in Drosophila, most organisms have several other
Skp1 family members, but in all cases their function
remain largely unknown (17). The sequence with high-
est homology to the human protein designated as
dSkpA (GadFly annotation CG16983) was found 76%
identical to hSkp1 whereas the other Drosophila Skp1
related sequences showed lower degrees of identity
(ranging between 17 and 67%). dSkpA is also the clos-
est Drosophila homologue of the yeast Skp1 protein
and the putative C. elegans Skp1 (Fig. 1). A similar
BLAST search was performed using the human Rbx1
protein sequence against the BDGP database and
three Drosophila sequences were found with signifi-
cant homology to the human protein. The sequence
corresponding to the GadFly annotation CG16982, des-
ignated as dRbx1, presents the highest degree of con-
servation with hRbx1 (91% identity) (18). The other
two Drosophila Rbx-related sequences are 70%
(CG16988) and 30% (CG18042) identical to hRbx1.
Nevertheless, CG18042 shows the highest identity
(70%) with the APC11 anaphase-promoting complex
subunit (19). In mammalian and yeast cells, the third
core component of SCF complexes is a member of the
cullin family, Cul1. In Drosophila, seven members of
this family can be found. The Drosophila orthologue of
human Cul1 has been recently described as a cell-cycle
regulator named dCul1 (11), which is 62% identical to
the human protein. Another Drosophila cullin family
member (GadFly annotation CG11861) is highly con-
served with human Cul3 (67% identity), a protein im-
plicated in cyclin E ubiquitination (20).
Once the components of a putative Drosophila SCF
complex were identified, we employed the yeast two-
hybrid system to investigate interactions between
FIG. 1. Multiple sequence alignment of dSkpA with its closest homologues in different species. Drosophila SkpA protein (dSkpA) was
aligned with human Skp1 (hSkp1, GenBank Acc. No. AAC50241), S. cerevisiae Skp1 (ScSkp1, GenBank Acc. No. AAB17500), and the
putative C. elegans Skp1 (CeSkp1, GenBank Acc. No. AAB17536) using the ClustalW1.8 program. dSkpA is 76% identical to hSkpA, 41%
identical to ScSkp1, and 35% identical to CeSkp1. Residues identical in all the sequences are shaded in black. Residues identical among three
of the four sequences are shaded in gray.
Vol. 286, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
359

4. them. For this purpose, fusion proteins corresponding
to dskpA, dcul1, dcul3, and drbx1, as well as the F-box
protein Slmb and three E2 ubiquitin conjugating en-
zymes were generated by linking either the yeast Gal4
DNA-binding domain or the Gal4 transcription activa-
tion domain to their N-terminal ends. As depicted in
Table 1, specific dCul1–dSkpA and dSkpA–Slmb inter-
actions were observed. The interaction between dCul1
and dSkpA was observed only when dSkpA was placed
as a bait and dCul1 as a prey. The direct interactions
dCul1–dSkpA and dSkpA–Slmb are consistent with
similar results in mammalian systems where Skp1
binds directly to Cdc53/hCul1 (5, 21, 22) and to ␤TRcP
(23). Interestingly, a second cullin tested, dCul3 failed
to interact with dSkpA and Slmb, confirming that
dCul1–dSkpA interaction is specific. Similar results
have been shown with human cullin family proteins
where only Cullin1, but not other closely related mem-
bers of the family, selectively interacts with human
Skp1 (22).
In order to increase sensitivity, the interactions
tested as negative in the plate assay were retested in
liquid assays for ␤-galactosidase activity. With this
assay, two new interactions were recorded as positive:
UbcD1-dCul1 and UbcD1-dCul3. Among the three Dro-
sophila E2s conjugating enzymes tested, only UbcD1
interacts with the cullins dCul1 and dCul3 while no
interactions were detected between this cullins and
UbcD6 or UbcD10, indicating that E2s are probably
not interchangeable. Multiple E2s have been reported
in diverse organisms, and these likely possess specific-
ity for different classes of target proteins (24). Drosoph-
ila UbcD1 appears to influence the degradation of a
wide array of proteins (12, 25–27) and can functionally
complement the yeast UBC4 mutant phenotype (12).
Consistently, UbcD1 gene shows the highest sequence
similarity to yeast Ubc4/Ubc5 (79% identity) and hu-
man UbcH5 (94% identity), and E2 enzyme implicated
in SCF␤-TrCP
-dependent degradation of I␬B (25). Several
other E2 enzymes exist in Drosophila showing lower
degrees of identity with UbcH5 (32 to 66%).
dRbx1-Gal4 DNA-binding domain fusion protein
(bait) rendered strong transcriptional activation even
in the absence of a prey. Similarly, dRbx1 placed as a
prey brought about activation in the absence of a bait.
Therefore, interactions with dRbx1 could not be as-
sessed by the two hybrid system and were evaluated in
vitro. A glutathione-S-transferase (GST)-tagged dRbx1
protein was prepared by fusing GST to its N-terminus
and expressed in bacteria. Interactions of GST–dRbx1
with different proteins generated by in vitro transcrip-
tion and translation (IVTT) were assessed by the GST
pull down assay. As shown in Fig. 2A, dRbx1 interacts
specifically with dCul1 and the F-box protein Slmb but
fails to interact with dSkpA. This is consistent with
results reported in yeast where Hrt1 binds Cdc53 (29)
and the F-box proteins Grr1 and Cdc4 (18). In accor-
dance with our result in Drosophila, no direct interac-
tion between Skp1 and Rbx1 was reported in any or-
ganism. dRbx1 did not show self-interaction in the
GST-pull down assay probably reflecting the fact that
it is present in the complex as a monomer (Fig. 2A).
Similar in vitro experiments were performed in order
to confirm the interactions assessed through the yeast
two-hybrid system. For this purpose His-tagged dSkpA
and UbcD1 proteins were generated by fusing six his-
tidine residues to the N-terminus of dSkpA and UbcD1.
Both proteins were expressed in bacteria and purified
using an iminodiacetic acid-sepharose column charged
with Ni2ϩ
. Figure 2B shows that radiolabeled Slmb and
dCul1, but not dRbx1, generated by IVTT specifically
interact with the His-dSkpA and His-UbcD1 chimeras.
These results confirm the ones obtained in the two-
hybrid system and add the observation of a direct in-
teraction between UbcD1 and Slmb. We observed that
Cul1 and Slmb migrate as a doublet (Fig. 2). Cul1
double band could be due to conjugation to endogenous
Nedd8/Rub1 present in the reticulocyte lysate. Nedd8/
Rub1 was shown to be covalently attached to yeast
Cdc53 and to all human cullin-family proteins result-
ing in appearance of a higher molecular mass polypep-
tide (30, 31). Slmb doublet could be due to some un-
known different posttranslational modification.
TABLE 1
Specific Interactions in the Two-Hybrid System
Gal4AD-hybrid
(pACT2)
Gal4BD-hybrid
(pAS2-1) His/Ade ␤-Gal
dSkpA dCul1 ϩ ϩ
dCul1 dSkpA Ϫ Ϫ
dSkpA dCul3 Ϫ Ϫ
dCul3 dSkpA Ϫ Ϫ
dSkpA dSkpA Ϫ Ϫ
dCul1 dCul1 Ϫ Ϫ
UbcD1 dCul1 Ϫ Ϫ
UbcD1 dCul3 Ϫ Ϫ
UbcD6 dCul1 Ϫ Ϫ
UbcD6 dCul3 Ϫ Ϫ
UbcD10 dCul1 Ϫ Ϫ
UbcD10 dCul3 Ϫ Ϫ
UbcD6 dSkpA Ϫ Ϫ
UbcD1 dSkpA Ϫ Ϫ
UbcD10 dSkpA Ϫ Ϫ
— dRbx1 ϩ ϩ
dRbx1 — ϩ ϩ
Slmb dCul1 Ϫ Ϫ
Slmb dCul3 Ϫ Ϫ
Slmb dSkpA ϩ ϩ
Note. Plasmids expressing the indicated fusion proteins were co-
transformed into yeast cells. Three independent colonies for each
transformation were streaked on SD-Leu-Trp-Ade-His selective me-
dium containing X-Gal. Interactions between fusion proteins were
assessed by growth on the selective medium, which indicates the
activation of the ADE2 and HIS3 reporter genes, and by their ability
to induce activation of the LacZ reporter gene recorded by blue color
staining.
Vol. 286, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
360

5. We next examined whether Drosophila dCul1,
dSkp1, and dRbx1 genes are able to functionally com-
plement the lethal phenotype of yeast strains lacking
cdc53, skp1, and hrt1, respectively. Tetrad analysis
of heterozygous disruptant strains CSLZ019(HE),
SWY100, and SWY200 was performed. For each tetrad
dissected, only two viable colonies were produced, both
of which were histidine auxotrophs. Consistent with
the essential nature of these genes, only hisϪ
spores
gave rise to normal size colonies, whereas inviable
spores formed microcolonies of 4–8 multibudded and
elongated cells.
Drosophila Rbx1 rescued the lethal phenotype of the
⌬hrt1 yeast strain. Tetrad analysis of strain SWY200
transformed with pYes2-dRbx1 yielded 2, 3, and 4 vi-
able spores on plates with galactose. Consistent with
this, hisϩ
viable spores were only obtained on galac-
tose, but not on glucose plates, and were always uraϩ
,
indicating that the lethal phenotype of ⌬hrt1 was sup-
presed by expression of Drosophila Rbx1 (Fig. 3A).
FIG. 2. (A) dRbx1 binds directly to dCul1 and Slmb. GST or GST-dRbx1 expressed in bacteria were bound to GSH-agarose beads and then
incubated with dSkpA, dCul1, dRbx1, or Slmb translated in a reticulocyte lysate in the presence of [35
S] methionine. Agarose beads bound
to GST-fusion proteins were precipitated and washed. Samples were dissolved in SDS-sample buffer and associated proteins were detected
by SDS–PAGE followed by fluorography. (B) dSkpA and UbcD1 bind directly to dCul1 and Slmb. UbcD1-His or dSkpA-His purified with
Niϩ
-iminodiacetic acid-sepharose beads were incubated with dCul1, dRbx1, or Slmb translated in a reticulocyte lysate in the presence of
[35
S] methionine. His-fusion proteins were then recovered by immunoprecipitation with anti-His antibody. Samples were dissolved in
SDS-sample buffer and associated proteins were detected by SDS–PAGE followed by fluorography.
Vol. 286, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
361

6. Haploid strains ⌬hrt1 rescued by pYes2-dRbx1 on
plates with galactose, grew on liquid medium contain-
ing galactose with division times comparable to those
of the wild-type strain. The ability of the comple-
mented strain to grow on liquid medium was strictly
dependent on the expression of the Drosophila gene.
When cultures of this strain that were growing expo-
nentially in liquid medium with galactose were shifted
to the same medium containing glucose, a rapid arrest
in growth was observed due to repression of the heter-
ologous gene (data not shown). In addition, repression
of Drosophila dRbx1 by growth in glucose caused the
yeast cells to exhibit the characteristic phenotype of
hrt1-deficient yeast strains (32) i.e., accumulation of
elongated cells with multiple abnormally shaped buds
(Fig. 3B). The ability of dRbx1 to complement ⌬hrt1
yeast phenotypes is similar to the reported complemen-
tation by the murine or human Rbx1 homologues (18,
33, 34) and demonstrates the functional conservation
of dRbx1 in Drosophila.
dCul1 and dSkpA failed to complement the lethal
phenotype of the corresponding null mutations in S.
cerevisiae although the complementation tests were
performed using constructs bearing the Drosophila
cDNAs under control of two different promoters in two
different yeast strains (see Materials and Methods).
The lack of functional complementation of the cdc53
strain may be due to the low level of identity between
the Drosophila and yeast proteins (28%). In accordance
with our data, hCul1 does not complement a cdc53 null
mutant yeast strain although it complements a
thermo-sensitive cdc53 mutation (21). On the other
hand, even though the overall conservation between
dSkpA and yeast Skp1 is higher (55% identity), the
recent crystal structure of a human Skp1 containing
complex suggests that divergences in its variable
C-terminal helix, involved in the recognition of specific
subsets of F-box proteins, could be responsible for the
lack of complementation (17). In this work, other mem-
bers of the Drosophila Skp1 family were not assayed
for the ability to interact with components of the SCF
complex or to complement the Skp1 yeast null mutant.
Thus, we can not rule out the possibility that one of
such proteins is the actual Skp1 functional homologue.
Our results, summarized in Fig. 4, provide evidences
that dRbx1, dSkpA, and dCul1 are components of a
Drosophila SCF complex containing the F-box protein
Slmb that functions in combination with the ubiquitin
conjugating enzyme UbcD1. Further characterization
of this SCF complex will require the demonstration of
its ubiquitin ligase activity.
ACKNOWLEDGMENTS
We thank all members of Wappner’s lab for critical reading of the
manuscript. This work was supported by a grant from Agencia Na-
FIG. 3. Drosophila Rbx1 complements the lethal phenotype of a
⌬hrt1 yeast strain. (A) Growth of two sister spores (wild-type and
⌬hrt1) derived from SWY200 bearing pYES2-dRbx1. Plates with
glucose or galactose were incubated at 30°C for 48 h. (B) Cell mor-
phology of ⌬hrt1 strains transformed with pYES2-dRbx1. Exponen-
tially growing yeast strains in liquid media containing galactose
were shifted to liquid media containing glucose for 8 h before pho-
tography.
FIG. 4. Schematic representation of a putative Drosophila SCF
complex as determined by direct interactions in the two-hybrid sys-
tem and in vitro assays.
Vol. 286, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
362

7. cional de Promocion Cientifica y Tecnologica (PICT 01-03390). S.N.B.
is a postdoctoral fellow from Consejo Nacional de Investigaciones
Cientificas y Tecnicas (CONICET). M.M. is a fellow of Proyecto
FOMEC. P.W. is member of the investigator career of CONICET.
REFERENCES
1. Hershko, A., and Ciechanover, A. (1998) The ubiquitin system.
Annu. Rev. Biochem. 67, 425–479.
2. Deshaies, R. J. (1999) SCF and Cullin/Ring H2-based ubiquitin
ligases. Annu. Rev. Cell Dev. Biol. 15, 435–467.
3. Patton, E. E., Willems, A. R., and Tyers, M. (1998) Combinatorial
control in ubiquitin-dependent proteolysis: Don’t Skp the F-box
hypothesis. Trends Genet. 14, 236–243.
4. Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J. W.,
and Elledge, S. J. (1996) SKP1 connects cell cycle regulators to
the ubiquitin proteolysis machinery through a novel motif, the
F-box. Cell 86, 263–274.
5. Skowyra, D., Craig, K. L., Tyers, M., Elledge, S. J., and Harper,
J. W. (1997) F-box proteins are receptors that recruit phosphor-
ylated substrates to the SCF ubiquitin-ligase complex. Cell 91,
209–219.
6. Maniatis, T. (1999) A ubiquitin ligase complex essential for the
NF-kappaB, Wnt/Wingless, and Hedgehog signaling pathways.
Genes Dev. 13, 505–510.
7. Theodosiou, N. A., Zhang, S., Wang, W. Y., and Xu, T. (1998)
Slimb coordinates wg and dpp expression in the dorsal-ventral
and anterior-posterior axes during limb development. Develop-
ment 125, 3411–3416.
8. Jian, J., and Struhl, G. (1998) Regulation of the Hedgehog and
Wingless signaling pathways by the F-box/WD40-repeat protein
Slimb. Nature 391, 493–496.
9. Spencer, E., Jiang, J., and Chen, Z. J. (1999) Signal-induced
ubiquitination of IkappaBalpha by the F-box protein Slimb/beta-
TrCP. Genes Dev. 13, 284–294.
10. Wojcik, E. J., Glover, D. M., and Hays, T. S. (2000) The SCF
ubiquitin ligase protein slimb regulates centrosome duplication
in Drosophila. Curr. Biol. 10, 1131–1134.
11. Filippov, V., Filippova, M., Sehnal, F., and Gill, S. S. (2000)
Temporal and spatial expression of the cell-cycle regulator cul1-
in Drosophila and its stimulation by radiation-induced apopto-
sis. J. Exp. Biol. 20, 2747–2756.
12. Treier, M., Seufert, W., and Jentsch, S. (1992) Drosophila UbcD1
encodes a highly conserved ubiquitin-conjugating enzyme in-
volved in selective protein degradation. EMBO J. 11, 367–372.
13. Koken, M. H., Reynolds, P., Jaspers-Dekker, I., Prakash, L.,
Prakash, S., Bootsma, D., and Hoeijmakers, J. H. (1991) Dhr6, a
Drosophila homolog of the yeast DNA-repair gene RAD6. Proc.
Natl. Acad. Sci. USA 88, 3832–3836.
14. Aguilera, M., Oliveros, M., Martinez-Padron, M., Barbas, J. A.,
and Ferrus, A. (2000) Ariadne-1. A vital Drosophila gene is
required in development and defines a new conserved family of
ring-finger proteins. Genetics 155, 1231–1244.
15. Wach, A., Brachat, A., Alberti-Segui, C., Rebischung, C., and
Philippsen, P. (1997) Heterologous HIS3 marker and GFP re-
porter modules for PCR-targeting in Saccharomyces cerevisiae.
Yeast 13, 1065–1075.
16. Mumberg, D., Muller, R., and Funk, M. (1995) Yeast vectors for
the controlled expression of heterologous proteins in different
genetic backgrounds. Gene 156, 119–122.
17. Schulman, B. A., Carrano, A. C., Jeffrey, P. D., Bowen, Z., Kin-
nucan, E. R., Finnin, M. S., Elledge, S. J., Harper, J. W., Pagano,
M., and Pavletich, N. P. (2000) Insights into SCF ubiquitin
ligases from the structure of the Skp1–Skp2 complex. Nature
408, 381–386.
18. Kamura, T., Conrad, M. N., Yan, Q., Conaway, R. C., and Con-
away, J. W. (1999) The Rbx1 subunit of SCF and VHL E3
ubiquitin ligase activates Rub1 modification of cullins Cdc53 and
Cul2. Genes Dev. 13, 2928–2933.
19. Ohta, T., Michel, J. J., Schottelius, A. J., and Xiong, Y. (1999)
ROC1, a homolog of APC11, represents a family of cullin part-
ners with an associated ubiquitin ligase activity. Mol. Cell 3,
535–541.
20. Singer, J. D., Gurian-West, M., Clurman, B., and Roberts, J. M.
(1999) Cullin-3 targets cyclin E for ubiquitination and controls S
phase in mammalian cells. Genes Dev. 13, 2375–2387.
21. Lyapina, S. A., Correl, C. C., Kipreos, E. T., and Deshaies, R. J.
(1998) Human CUL1 forms an evolutionarily conserved ubiq-
uitin ligase complex (SCF) with SKP1 and an F-box protein.
Proc. Natl. Acad. Sci. USA 95, 7451–7456.
22. Michel, J. J., and Xiong, Y. (1998) Human CUL-1, but not
other cullin family members, selectively interacts with SKP1
to form a complex with SKP2 and cyclin A. Cell Growth Diff.
9, 435–449.
23. Winston, J. T., Strack, P., Beer-Romero, P., Chu, C. Y., Elledge,
S. J., and Harper, J. W. (1999) The SCFbeta-TRCP-ubiquitin
ligase complex associates specifically with phosphorylated de-
struction motifs in IkappaBalpha and beta-catenin and stimu-
lates IkappaBalpha ubiquitination in vitro. Genes Dev. 13, 270–
283.
24. Oughtred, R., Bedard, N., Vrielink, A., and Wing, S. S. (1998)
Identification of amino acid residues in a class I ubiquitin-
conjugating enzyme involved in determining specificity of con-
jugation of ubiquitin to proteins. J. Biol. Chem. 273, 18435–
18442.
25. Cenci, G., Rawson, R. B., Belloni, G., Castrillon, D. H., Tudor,
M., Petrucci, R., Goldberg, M. L., Wasserman, S. A., and Gatti,
M. (1997) UbcD1, a Drosophila ubiquitin-conjugating enzyme
required for proper telomere behavior. Genes Dev. 11, 863–
875.
26. Tang, A. H., Neufeld, T. P., Kwan, E., and Rubin, G. M. (1997)
PHYL acts to down-regulate TTK88, a transcriptional repressor
of neuronal cell fates, by a SINA-dependent mechanism. Cell 90,
459–467.
27. Lilly, M. A., de Cuevas, M., and Spradling, A. C. (2000) Cyclin A
associates with the fusome during germline cyst formation in the
Drosophila ovary. Dev. Biol. 218, 53–63.
28. Gonen, H., Bercovich, B., Orian, A., Carrano, A., Takizawa, C.,
Yamanaka, K., Pagano, M., Iwai, K., and Ciechanover, A. (1999)
Identification of the ubiquitin carrier proteins, E2s, involved
in signal-induced conjugation and subsequent degradation of
IkappaBalpha. J. Biol. Chem. 274, 14823–14830.
29. Kamura, T., Koepp, D. M., Conrad, M. N., Skowyra, D., More-
land, R. J., Iliopoulos, O., Lane, W. S., Kaelin, W. G. Jr., Elledge,
S. J., Conaway, R. C., Harper, J. W., and Conaway, J. W. (1999)
Rbx1, a component of the VHL tumor suppressor complex and
SCF ubiquitin ligase. Science 284, 657–661.
30. Lammer, D., Mathias, N., Laplaza, J. M., Jiang, W., Liu, Y.,
Callis, J., Goebl, M., and Estelle, M. (1998) Modification of yeast
Cdc53p by the ubiquitin-related protein rub1p affects function of
the SCFCdc4 complex. Genes Dev. 12, 914–926.
31. Liakopoulos, D., Doenges, G., Matuschewski, K., and Jentsch, S.
(1998) A novel protein modification pathway related to the ubiq-
uitin system. EMBO J. 17, 2208–2214.
32. Blondel, M., Galan, J. M., and Peter, M. (2000) Isolation and
characterization of HRT1 using a genetic screen for mutants
unable to degrade Gic2p in Saccharomyces cerevisiae. Genetics
155, 1033–1044.
33. Seol, J. H., Feldman, R. M., Zachariae, W., Shevchenko, A.,
Correll, C. C., Lyapina, S., Chi, Y., Galova, M., Claypool, J.,
Vol. 286, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
363

8. Sandmeyer, S., Nasmyth, K., Deshaies, R. J., Shevchenko, A.,
and Deshaies, R. J. (1999) Cdc53/cullin and the essential Hrt1
RING-H2 subunit of SCF define a ubiquitin ligase module that
activates the E2 enzyme Cdc34. Genes Dev. 13, 1614–1626.
34. Skowyra, D., Koepp, D. M., Kamura, T., Conrad, M. N., Con-
away, R. C., Conaway, J. W., Elledge, S. J., and Harper, J. W.
(1999) Reconstitution of G1 cyclin ubiquitination with complexes
containing SCFGrr1 and Rbx1. Science 284, 662–665.
Vol. 286, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
364