-
IntroductionThe septins are a family of filament-forming,
GTP-bindingproteins that were first discovered in yeast but are now
knownto be widely distributed and perhaps ubiquitous in the
fungiand animals (Field and Kellogg, 1999; Kartmann and Roth,2001;
Longtine et al., 1996; Longtine and Pringle, 1999;Momany et al.,
2001; Trimble, 1999). In some cell types(Hartwell, 1971; Kinoshita
et al., 1997; Neufeld and Rubin,1994), although apparently not in
all (Adam et al., 2000;Longtine et al., 1996; Nguyen et al., 2000),
septins are essentialfor cytokinesis. In addition, the yeast
septins play a variety ofother roles in cell surface organization;
in many cases, theseptins appear to serve as a scaffold or template
for otherproteins (Barral et al., 2000; Field and Kellogg, 1999;
Longtineand Pringle, 1999; Longtine et al., 2000; Takizawa et al.,
2000).Genetic, biochemical, and protein-localization data
suggestthat the septins probably have important non-cytokinesis
rolesin animal cells as well (Field and Kellogg, 1999; Kartmann
andRoth, 2001; Kinoshita et al., 2000; Larisch et al.,
2000;Longtine et al., 1996). Drosophila melanogasterhas at
leastfive septins, named Pnut, Sep1, Sep2, Sep4 and Sep5,
whosefunctions are not yet well understood (Adam et al., 2000;
Fares
et al., 1995; Field et al., 1996; Longtine et al., 1996;
Neufeldand Rubin, 1994). Progress will presumably depend, in
part,on identifying the proteins with which the septins
interact.
Here we used the yeast two-hybrid system to identify
proteinsthat interact with Drosophilaseptins. Among the positive
clonesobtained were ones encoding the Drosophila homologues ofyeast
Uba2p and Ubc9p, which catalyze the activation andconjugation,
respectively, of the ubiquitin-like protein Smt3p(Johnson and
Blobel, 1997; Johnson et al., 1997; Schwarz et al.,1998). Smt3p is
one of several ubiquitin-like proteins recentlyidentified and shown
to become covalently linked to otherproteins as a form of
post-translational modification(Hochstrasser, 2000; Melchior,
2000). For example, themammalian Smt3p homologue SUMO-1 modifies
RanGAP1(Mahajan et al., 1997; Matunis et al., 1996; Matunis et al.,
1998),several transcriptional regulators including p53 and
c-Jun(Gostissa et al., 1999; Muller et al., 2000; Rodriguez et
al.,1999), and the RING-finger protein Mdm2 (Buschmann et
al.,2000). Yeast Smt3p appears to modify multiple nuclear
proteins(Johnson and Blobel, 1999; Meluh and Koshland,
1995),consistent with the observations that Uba2p and Ubc9p
localizepredominantly in the nucleus (Dohmen et al., 1995; Seufert
etal., 1995). Importantly, it was recently shown that several
yeast
1259
The septins are a family of proteins involved in cytokinesisand
other aspects of cell-cortex organization. In a two-hybrid screen
designed to identify septin-interactingproteins in Drosophila, we
isolated several genes, includinghomologues (Dmuba2 and Dmubc9) of
yeast UBA2 andUBC9. Yeast Uba2p and Ubc9p are involved in
theactivation and conjugation, respectively, of the ubiquitin-like
protein Smt3p/SUMO, which becomes conjugated to avariety of
proteins through this pathway. Uba2p functionstogether with a
second protein, Aos1p. We also cloned andcharacterized the
Drosophila homologues of AOS1(Dmaos1) and SMT3 (Dmsmt3). Our
biochemical datasuggest that DmUba2/DmAos1 and DmUbc9 indeed act
asactivating and conjugating enzymes for DmSmt3, implyingthat this
protein-conjugation pathway is well conserved inDrosophila.
Immunofluorescence studies showed thatDmUba2 shuttles between the
embryonic cortex and nuclei
during the syncytial blastoderm stage. In older embryos,DmUba2
and DmSmt3 are both concentrated in the nucleiduring interphase but
dispersed throughout the cellsduring mitosis, with DmSmt3 also
enriched on thechromosomes during mitosis. These data suggest
thatDmSmt3 could modify target proteins both inside andoutside the
nuclei. We did not observe any concentration ofDmUba2 at sites
where the septins are concentrated, andwe could not detect DmSmt3
modification of the threeDrosophila septins tested. However, we did
observeDmSmt3 localization to the midbody during cytokinesisboth in
tissue-culture cells and in embryonic mitoticdomains, suggesting
that DmSmt3 modification of septinsand/or other midzone proteins
occurs during cytokinesis inDrosophila.
Key words: Drosophila, Septins, Ubiquitin-like proteins,
Cytokinesis
Summary
Identification of septin-interacting proteins
andcharacterization of the Smt3/SUMO-conjugationsystem in
DrosophilaHsin-Pei Shih 1,*, Karen G. Hales 1,§, John R. Pringle
1,2,3 and Mark Peifer 1,31Department of Biology, 2Program in
Molecular Biology and Biotechnology and 3Lineberger Comprehensive
Cancer Center, University of NorthCarolina, Chapel Hill, NC 27599
USA*Present address: Institute of Neuroscience, Howard Hughes
Medical Institute, University of Oregon, Eugene, OR 97403
USA§Present address: Department of Biology, Davidson College,
Davidson, NC 28035, USAAuthors for correspondence (e-mail:
[email protected]; [email protected])
Accepted 13 December 2001Journal of Cell Science 115, 1259-1271
(2002) © The Company of Biologists Ltd
Research Article
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1260
septins are modified by Smt3p (Johnson and Blobel,
1999;Takahashi et al., 1999) (P. Meluh, personal
communication).
The functions of modification by Smt3/SUMO and
otherubiquitin-like proteins are not entirely clear, but they
appearto include regulation of protein localization
(RanGAP1),transcriptional activity (p53 and c-Jun), and protein
stability(Mdm2). Unlike ubiquitin itself (Hochstrasser, 1996),
otherubiquitin-like proteins do not appear to target modified
proteinsfor degradation and in fact may stabilize target proteins
bypreventing their ubiquitination (Hochstrasser, 2000;
Melchior,2000).
The ubiquitin-like proteins have differing and generally
lowlevels of sequence similarity to ubiquitin itself; for
example,yeast Smt3p and ubiquitin have only 17% identity in
theiramino acid sequences. Nonetheless, the respective
conjugationpathways have many common features (Hochstrasser,
2000;Melchior, 2000). In most cases, ultimate conjugation is by
anisopeptide bond between a C-terminal glycine on ubiquitin orthe
ubiquitin-like protein and the ε-amino group of a lysine inthe
target protein. The conjugation reaction involves an
‘E2’conjugating enzyme and, at least in some cases, an ‘E3’
proteinligase involved in target-protein recognition. The
conjugatingenzyme receives the ubiquitin or ubiquitin-like protein
from an‘E1’ activating enzyme, which first adenylates the
C-terminusof ubiquitin or the ubiquitin-like protein and then links
it by athiolester bond to a cysteine in the E1 enzyme. The
enzymaticmechanisms of the various E1 enzymes are similar, and
theenzymes themselves are structurally related; this is also true
ofthe various E2 enzymes. In the case of yeast Smt3p, activationis
carried out by a heterodimer of Uba2p and a second protein,Aos1p,
and links the C-terminus of Smt3p to the active sitecysteine (Cys
177) in Uba2p (Johnson et al., 1997). Smt3p isthen transferred and
conjugated by another thiolester bond toCys 93 in Ubc9p (Johnson
and Blobel, 1997; Schwarz et al.,1998). The available data suggest
that the same enzymaticmachinery is used for SUMO-1 conjugation in
mammaliancells (Desterro et al., 1997; Desterro et al., 1999;
Hochstrasser,2000; Melchior, 2000; Okuma et al., 1999). Although
E3enzymes have not yet been identified for most
ubiquitin-likeproteins (Hochstrasser, 2000; Melchior, 2000), recent
studieshave identified two possible E3 enzymes for
Smt3/SUMOmodification (Johnson and Gupta, 2001; Kahyo et al.,
2001;Takahashi et al., 2001).
In this study, we sought septin-interacting proteins
inDrosophilaand attempted to elucidate the relationship betweenthe
septins and the Smt3 conjugation system in this organism.In
addition, we used biochemical and cell biologicalapproaches to
characterize the Drosophila Smt3 conjugationpathway and investigate
its possible functions.
Materials and MethodsStrains and molecular biology methodsThe
wild-type Canton S strain of D. melanogasterand the
Clone-8Drosophila cultured cell line (Peel and Milner, 1992) were
used. S.cerevisiaestrains EGY48R (α his3 trp1 ura3-52
leu2::pLEU2-lexAop6[pSH18-34]) (DeMarini et al., 1997) and JD90-1A
(α his3 leu2 lys2trp1 ura3 uba2∆::HIS3) containing the uba2ts10
plasmid pIS2-ts10(Johnson et al., 1997) were used for two-hybrid
analyses and forexpressing Dmuba2, respectively, and were grown
under standardconditions (Guthrie and Fink, 1991). Escherichia
colistrains DH5α andDH12S (Life Technologies, Grand Island, NY)
were used for routine
plasmid propagation, and strains JMB9r–m+∆trpF (Sterner et al.,
1995)and JM109 (Promega, Madison, WI) were used as described
below.
Standard recombinant DNA techniques (Ausubel et al., 1994;
Gietzet al., 1992) were used except where noted. PCR used either
theExpand High Fidelity kit (Boehringer Mannheim, Indianapolis, IN)
togenerate fragments for cloning into fusion-protein and
two-hybridvectors or Taq DNA polymerase (Promega) for other
applications.Oligonucleotide primers were obtained from Integrated
DNATechnologies (Coralville, IA).
Two-hybrid assays and screeningTwo-hybrid assays were performed
as described previously (DeMariniet al., 1997; Fields and
Sternglanz, 1994; Gyuris et al., 1993) using theLexA DNA-binding
domain (DBD) plasmid pEG202 (Ausubel et al.,1994) and activation
domain (AD) plasmid pJG4-5 (Ausubel et al., 1994)or pJG4-5PL
(DeMarini et al., 1997). The baits used for screeningcontained
full-length sequences of pnut, sep1, and sep2(J. C. Adam etal.,
unpublished). The libraries used (provided by R. Finley,
HarvardMedical School, Boston, MA) were RFLY1, a
DrosophilaembryoniccDNA library, and RFLY5, an imaginal-disc cDNA
library, both inpJG4-5. Each bait plasmid was co-transformed into
yeast strainEGY48R with each of the two libraries. Transformants
were plated ontoSynthetic Minimal plates (Guthrie and Fink, 1991)
containing 2%galactose + 1% raffinose and grown for 3-6 days to
select Leu+ clones,which were further evaluated using both filter
and liquid-culture β-galactosidase assays (Ausubel et al., 1994).
Plasmids from positiveclones were rescued into E. coli strain JMB9
and retested by co-transformation into EGY48R together with one of
the pEG202-basedplasmids and measurement of β-galactosidase
activity. Inserts frompositive plasmids were then sequenced. We
screened >106 transformantsfor each library with each bait.
Additional two-hybrid tests used the following plasmids.
pJG4-5PL,expressing full-length anillin, was provided by Julie
Brill (Hospital forSick Children, Toronto, Canada). Construction of
plasmids expressingN- or C-terminal fragments of Pnut (Pnut-N,
amino acids 1-426; Pnut-C, amino acids 407-539), Sep1 (Sep1-N,
amino acids 1-325; Sep1-C,amino acids 306-361), or Sep2 (Sep2-N,
amino acids 1-337; Sep2-C,amino acids 318-419) in pEG202 will be
described in detail elsewhere(J. C. Adam et al., unpublished).
Full-length Dmuba2and Dmaos1andthe 5′ (amino acids 1-350) and 3′
(amino acids 351-700) halves ofDmuba2were amplified by PCR using
the cloned genes (see below) astemplates and the primers shown in
Table 1. The amplified Dmuba2and its fragments were cloned into
pEG202 and pJG4-5PL at theirEcoRI sites, andDmaos1was cloned into
pEG202 and pJG4-5PL attheir XhoI sites. The full-length genes and
the Dmuba2C-terminalconstructs contain the original stop codons,
whereas the Dmuba2N-terminal constructs use a stop codon in the
vector downstream of thepolylinker.
Isolation of full-length Dmuba2, Dmaos1, and Dmsmt3 clonesTwo
independent positive clones from the two-hybrid screen
encodedC-terminal fragments (including the putative stop codon) of
the samegene. To isolate the 5′ end of this gene, we identified an
EST clone(LD03967) from the Berkeley DrosophilaGenome Project
(BDGP)that extended further in the 5′ direction and then performed
PCR asdescribed previously (McCurdy and Kim, 1998), using a
Schneidercell cDNA library in vector pDB20 (Becker et al., 1991) as
templateand primers (Table 1) that corresponded to vector sequences
and tothe 5′ end of the LD03967 sequences, respectively. In
aggregate, thecDNA sequences revealed an apparently complete ORF of
2,100 bpthat had similarity to yeast UBA2 (see Results).
Full-length Dmuba2was then cloned by PCR using the primers shown in
Table 1, theSchneider cell cDNA library as the template, and
plasmid pGEM-T(Promega). We mapped Dmuba2 to position 66B6-66B10
onchromosome arm 3L using a high-density filter of P1 clones
(Genome
Journal of Cell Science 115 (6)
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1261Smt3 conjugation system in Drosophila
Systems, St. Louis, MO), as subsequently confirmed by data from
thegenome project (see FlyBase).
Full-length clones of Dmaos1 and Dmsmt3 were obtained
byidentifying and sequencing BDGP EST clones (GM10027 andGM01812)
that contained apparently complete ORFs with similarityto yeast
Aos1p and Smt3p, respectively (see Results).
Antibodies, immunoblotting, and immunoprecipitationRabbit
polyclonal anti-DmUbc9 antibodies (Joanisse et al., 1998)were
provided by Robert Tanguay (University of Laval, Quebec,Canada),
mouse monoclonal anti-Pnut antibody 4C9 (Neufeld andRubin, 1994)
and anti-myc antibody 9E10 (Evan et al., 1985) wereobtained from
the Developmental Studies Hybridoma Bank(University of Iowa, Iowa
City, IA), and anti-β-tubulin antibody wasobtained from
Amersham-Pharmacia Biotech (Piscataway, NJ).
To prepare DmUba2-specific antibodies, DNA encoding DmUba2amino
acids 553-700 was amplified by PCR using one of the two-hybrid
clones as the template and the primers shown in Table 1.
Theproducts were cut with EcoRI and cloned into pGEX-1
(Amersham-Pharmacia), producing pGEX-DmUba2, and into pMAL-c2
(NewEngland Biolabs, Beverly, MA), producing pMAL-DmUba2.
Theresulting glutathione-S-transferase (GST)- and
maltose-bindingprotein (MBP)-fusion proteins were expressed,
purified, and used toraise antibodies in rabbits by standard
methods (Ausubel et al., 1994)(Cocalico Biologicals, Reamstown,
PA). After five boosts, antibodies
raised against GST-DmUba2 were affinity purified using
MBP-DmUba2 that had been subjected to SDS-PAGE and blotted
tonitrocellulose (Pringle et al., 1989).
To prepare DmSmt3-specific antibodies, a His6-tagged
DmSmt3protein was generated. Dmsmt3codons 1-88 were amplified by
PCRusing Canton S genomic DNA (Sullivan et al., 2000) as the
templateand the primers shown in Table 1. The resulting product and
plasmidpQE30 (Qiagen) were digested with BamHI and HindIII and
ligatedtogether to produce pQE30-DmSmt3, which encodes DmSmt3
taggedat its N-terminus with His6 and truncated after the two
glycines thatpresumably represent the C-terminus of the mature
endogenousprotein (Johnson et al., 1997; Kamitani et al., 1997).
His6DmSmt3was expressed in strain JM109 and purified on Ni-NTA
columns asrecommended by Qiagen, and rabbit antibodies were raised
usingstandard protocols (Cocalico Biologicals). After four
boosts,DmSmt3-specific antibodies were affinity purified using
His6DmSmt3that had been subjected to SDS-PAGE and blotted to
nitrocellulose(see above).
For immunoblotting, samples were diluted or resuspended in 5×-
or2×-concentrated Laemmli buffer to achieve a 1× final
concentration,treated at 100°C for 5 minutes (except where noted),
analyzed by SDS-PAGE, and blotted to nitrocellulose. Blots were
blocked for 1 hour at23°C with 5% non-fat dry milk in TBS (Ausubel
et al., 1994) buffercontaining 0.1% Tween-20 (TBS-T), incubated
with primary antibodiesin the same buffer for 1 hour at 23°C,
washed three times in TBS-T at23°C (10 minutes per wash), incubated
with secondary antibodies inTBS-T containing 5% non-fat dry milk
for 1 hour at 23°C, and washedthree times in TBS-T at 23°C (5
minutes per wash). Proteins were thendetected using the enhanced
chemiluminescence (ECL) kit (Amersham-Pharmacia). The primary
antibodies used were purified anti-DmUba2(at 1:1500), anti-DmUbc9
(at 1:2000), and purified anti-DmSmt3 (at1:750). The secondary
antibody was HRP-conjugated goat anti-rabbit-IgG (at 1:5,000; ICN
Pharmaceuticals, Costa Mesa, CA). Fly extractswere prepared from
0-16-hour-old embryos as described previously (Paiet al., 1996)
using NET buffer (50 mM Tris-HCl, pH 7.5, 400 mM NaCl,5 mM EDTA, 1%
NP-40) containing phosphatase and proteaseinhibitors. In one case,
the buffer also contained 20 mM N-ethylmaleimide (NEM; Sigma).
To express Dmuba2 in yeast under GAL-promoter control,
weamplified full-length Dmuba2by PCR using the cloned cDNA
astemplate and the primers shown in Table 1, cut the product with
SalI,and cloned it into YCpIF2 (Foreman and Davis, 1994),
producingYCpIF2-DmUba2. Strain JD90-1A [pIS2-ts10] (see above)
wastransformed with YCpIF2-DmUba2 and grown under
conditionsselective for both plasmids and inducing or noninducing
for the GALpromoter. Yeast extracts were then prepared as described
previously(Ausubel et al., 1994).
To prepare fly extracts for immunoprecipitation (IP),
0-16-hour-oldembryos were collected, dechorionated, and rinsed as
describedpreviously (Pai et al., 1996). 100 µl of embryos were
homogenized at0°C in extraction buffer (500 µl of CER I plus 27.5
µl of CER II; bothreagents from the NE-PER kit, Pierce, Rockford,
IL). The extract wasadded to 1 ml of NET buffer containing
phosphatase and proteaseinhibitors (Pai et al., 1996) and
centrifuged for 10 minutes at 15,000 gin a microfuge at 4°C, and
the supernatant was collected. IPs werecarried out as described
previously (Peifer, 1993) using purified anti-DmUba2 (at 1:75),
anti-DmUbc9 (at 1:200), or monoclonal anti-myc9E10 (at 1:20). IPs
were analyzed by SDS-PAGE and immunoblotting.
In vitro protein-binding assaysCodons 1-76 of a Drosophila
ubiquitin gene (Lee et al., 1988) wereamplified by PCR using the
Schneider cell cDNA library as thetemplate and the primers shown in
Table 1. The resulting fragmentwas then cloned into pQE30 using
KpnI and HindIII. The resultingplasmid encodes ubiquitin (DmUb)
tagged at its N-terminus with His6and truncated after the Gly-Gly
that presumably represents the C-
Table 1. Oligonucleotide primers used in this study Name
Sequencea
Dmuba2NFb,c 5′-AGGAATTCATGGCAGCAGCTATCAATGGT-3′Dmuba2CRb,d
5′-ACGAATTCTTAATCGATACTGATGACTGC-3′Dmaos1Fe
5′-ACCTCGAGATGGTCGTGGATATGGACAC-3′Dmaos1Re
5′-ATCTCGAGTCACTTCGCTCCGATGGCCT-3′Dmuba2NRc
5′-AGGAATTCGAGTTTCAGGAAGTTAGCGCT-3′Dmuba2CFd
5′-ATGAATTCGAAGGCGACGATACTCTGGCT-3′ADH-Rf
5′-CTACAGGAAAGAGTTACTCAAG-3′PDmuba2O5f
5′-CTTGCGGGTCTTCCACAAAT-3′Dmuba2CFg
5′-AGAATTCGGATCCCTCTAAAGGGCGGCGTCACT-3′Dmuba2Cg
5′-GCAAGCTTTTAATCGATACTGATGACT-3′Dmuba2Rh
5′-CGAATTCGGATCCTATTAATCGATACT-3′Dmuba2JGh
5′-ATTAGAATTCGGCACGAGGCGGCG-3′Dmsmt3Fi
5′-ACGACGGGATCCGCATCGATGTCTGACGAAAAG-
AAGGGAGG-3′Dmsmt3Ri 5′-ACGACGAAGCTTGGCTGCAGGTCAAGCGCCACC-
AGTCTGCTGCTGG-3′Dmuba2FSj
5′-ATGTCGACATGGCAGCAGCTATCAATGGT-3′Dmuba2RSj
5′-ATGTCGACTTAATCGATACTGATGACTGC-3′DubiFk
5′-AGGTACCATGCAGATCTTTGTGAA-3′DubiRk
5′-ATAAGCTTTTATCCTCCACGGAGACGGAGCAC-3′
aRestriction sites used in cloning PCR products are
underlined.bPrimers used to amplify full-length Dmuba2for cloning
into pEG202 and
pJG4-5PL.cPrimers used to amplify the 5′ half of Dmuba2for
cloning into pEG202
and pJG4-5PL.dPrimers used to amplify the 3′ half of Dmuba2for
cloning into pEG202
and pJG4-5PL.ePrimers used to amplify full-length Dmaos1for
cloning into pEG202 and
pJG4-5PL.fPrimers used to amplify the 5′ end of Dmuba2from the
library in pDB20.gPrimers used to amplify full-length Dmuba2,
including 96 bp of 5′-UTR
but no 3′-UTR, for cloning into pGEM-T.hPrimers used to amplify
Dmuba2codons 553-700 for cloning into pGEX-
1 and pMAL-c2.iPrimers used to amplify codons 1-88 of Dmsmt3for
cloning into pQE30.jPrimers used to amplify the full-length
Dmuba2ORF (without flanking
sequences) for cloning into YCpIF2.kPrimers used to amplify
codons 1-76 of a Drosophilaubiquitin gene for
cloning into pQE30.
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1262
terminus of the mature endogenous protein. His6-DmSmt3 (see
above)and His6-DmUb were expressed separately in strain JM109
asrecommended by Qiagen and purified using the B-PER kit
(Pierce).Fly embryo extracts were prepared as described above
except thathomogenization was in 1 ml of RIPA buffer (50 mM
Tris-HCl, pH8.5, 300 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS,
1% NP-40) containing 20 mM imidazole, 2 mM ATP, 5 mM MgCl2, 0.1
mMDTT, 50 mM NaF, and protease inhibitors (see above). Extract
wascentrifuged for 10 minutes at 8000 g in a microfuge at 4°C, and
5 µgof His6-DmSmt3 or His6-DmUb was added to 450 µl of
supernatant.After incubation at 23°C for 15 minutes, 15 µl of
Ni-NTA beads wereadded, and the mixture was incubated at 4°C for 45
minutes. Thebeads were washed four times in RIPA containing 20 mM
imidazole,50 mM NaF, and protease inhibitors (as above). For
analysis underreducing conditions, beads were suspended in standard
1× Laemmlibuffer and analyzed by immunoblotting as described above.
Foranalysis under nonreducing conditions, beads were suspended in
1×Laemmli buffer without β-mercaptoethanol and heated at 68°C for
10minutes prior to SDS-PAGE and immunoblotting.
Immunofluorescence microscopyEmbryos collected after development
at 25°C were washed anddechorionated as described above, then
fixed, devitellinized, andstained as described (Cox et al., 1996).
The ages of syncytial-blastoderm embryos were determined by
counting the nuclei inlongitudinal sections. Standard morphological
criteria (Roberts, 1986)were used to identify other developmental
stages. Clone-8 cells werewashed three times with PBS, fixed with
2% paraformaldehyde inPBS for 10-15 minutes at 23°C, washed twice
with PBS, treated withprimary antibodies in Solution H (PBS
containing 0.1% Triton X-100and 1% normal goat serum) for 1 hour at
23°C, washed once withPBS, treated with secondary antibodies in
Solution H for 1 hour at23°C, and washed twice again with PBS. The
primary antibodies usedwere purified anti-DmUba2 (at 1:50) and
anti-DmSmt3 (at 1:75),
monoclonal anti-Pnut antibody 4C9 (at 1:3), and anti-β-tubulin
(at1:100). FITC- and rhodamine-conjugated goat anti-rabbit-IgG
andgoat anti-mouse-IgG (Jackson ImmunoResearch, West Grove, PA)were
used at 1:500; Alexa Fluor 488-conjugated goat
anti-rabbit-IgG(Molecular Probes, Eugene, OR) and Cy3-conjugated
goat anti-mouse-IgG (Jackson ImmunoResearch) were used at
1:1000.Embryos and cells were mounted in AquaPolymount
(Polysciences,Warrington, PA), then observed and photographed using
a Zeiss LSM410 confocal microscope.
ResultsIdentification of genes by two-hybrid interactions
withDrosophila septinsTo identify proteins interacting with the
Drosophilaseptins, weconducted two-hybrid screens using Pnut, Sep1,
and Sep2 asbaits (see Materials and Methods). These screens
identified 27positive clones that proved to represent eight genes
(Table 2).Among these were the other septins, as expected from
otherdata indicating that septins interact with one another
(seeDiscussion). In addition, the screens identified
Drosophilahomologues (Dmuba2and Dmubc9) of yeast UBA2and UBC9,whose
products are involved in the activation and conjugationof the
ubiquitin-like molecule Smt3p/SUMO (seeIntroduction). These
screening results and the recent discoveryof Smt3p modification of
septins in S. cerevisiae(Johnson andBlobel, 1999; Takahashi et al.,
1999) (P. Meluh, personalcommunication) stimulated us to study the
Smt3p/SUMOconjugation machinery in Drosophila.
In further two-hybrid analyses, the C-terminal portion ofDmUba2
interacted strongly with full-length Sep1 and Sep2 andwith the
N-terminal portion of Sep1. Interactions were alsodetected with the
N-terminal portions of Pnut and Sep2 and with
Journal of Cell Science 115 (6)
Table 2. Two-hybrid interactions of Drosophilaseptins with each
other, sumoylation enzymes, and other proteinsa
AD Fusion
DBD fusion Pnutb Sep2c DmUba2d DmUba2e DmUbc9f Sip1g Sip2g Sip3g
Sip4h
Pnut-F 526, 1058 79-218 16 15 25 347, 445 14 13 18Pnut-N − − 334
20 181 37 14 16 120Pnut-C − − 39 17 44 5474 20 22 39Sep1-F − 1821
1122, 1994 22 636, 1749 28 295 19 618, 601Sep1-N − − 2621 27 2550
29 94 9 530Sep1-C − − 168 24 99 62 56 20 278Sep2-F 126-354 − 600,
570 24 765 38 413, 509 758, 464 1048Sep2-N − − 350 29 830 41 108 16
184Sep2-C − − 193 45 169 52 107 2632 517
aNumbers indicate units of β-galactosidase activity. Those in
bold face represent values obtained in the original screens; others
were derived from thesubsequent systematic analyses. For the
DBD-septin fusions, F denotes the full-length septins, N denotes
fragments extending from the N-terminus to the N-terminal boundary
of the predicted coiled-coil domains, and C denotes C-terminal
fragments consisting essentially of the predicted coiled-coil
domains. A full-length AD-anillin clone (see Materials and Methods)
and the empty vector pJG4-5 were also used as negative controls
with each of the DBD fusions; in all cases,these yielded low values
similar to those obtained with the full-length DmUba2.
b10 positive clones from the screens proved to contain fragments
of pnut. The two isolated with Pnut as bait contained sequences
beginning at amino acids 78and 142 but were not completely
sequenced. All of the eight isolated with Sep2 as bait contained
sequences beginning between amino acids 413 and 431 (of
the539-amino-acid protein) but were not completely sequenced.
cSeven positive clones from the screens proved to contain
fragments of sep2. All of the six isolated with Pnut as bait
contained sequences beginning betweenamino acids 265 and 288. In at
least two of these, the sequences appeared to run to the
C-terminus; the others were not completely sequenced. The clone
isolatedusing Sep1 as bait contained sequences starting at amino
acid 27 but was not completely sequenced.
dThree positive clones from the screens proved to contain
fragments of Dmuba2. Two of these (one from the Sep1 screen and one
from the Sep2 screen;interaction values shown in the table) encoded
amino acids 552-700; one of these clones was used in the systematic
two-hybrid analyses. The third Dmuba2clone(from the Sep1 screen;
708 units of β-galactosidase activity) encoded amino acids
427-700.
eFull-length AD-DmUba2 (see Materials and Methods and Table 3).
fThe single Dmubc9clone isolated in the screen contained the
complete ORF (see text). gEach of these interactors was represented
by a single positive clone from the screen.hThree positive clones
from the Sep1 screen contained identical fragments of the gene
designated sip4. The original interaction value for one of these
clones is
shown. This same AD-fusion clone was used in the systematic
two-hybrid analyses.
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1263Smt3 conjugation system in Drosophila
the C-terminal portions of Sep1 and Sep2 (Table 2). In
contrast,a full-length AD-DmUba2 fusion showed none of
theseinteractions (Table 2), although other studies (see Table
3)indicated that this fusion was functional for other
interactions.Interestingly, full-length AD-DmUbc9 showed a pattern
ofinteractions very similar to those seen with the
C-terminalportion of DmUba2.
The other genes identified in the screens had not beendescribed
previously; we designated them sip1-sip4 (for septin-interacting
protein). sip1(Accession No. AF221101; Drosophilagenome annotation
No. CG7238) encodes a protein withpredicted P-loop and coiled-coil
domains; it appeared to interactspecifically with the C-terminal
portion of Pnut (Table 2). sip2-sip4encode proteins without
obviously informative motifs. sip2(CG9188) encodes a protein that
interacted with full-length Sep1and Sep2 but not with Pnut. sip3
(CG1937) encodes a proteinthat appeared to interact specifically
with the C-terminal portionof Sep2. sip4 was identified
independently as dip2 (Dorsalinteracting protein 2) (Bhaskar et
al., 2000); it encodes a proteinthat interacted with all of the
Sep1 and Sep2 fusions and (weakly)with the N-terminal portion of
Pnut (Table 2).
Cloning and sequence analysis of Dmuba2 and Dmubc9We obtained a
full-length clone of Dmuba2as described in theMaterials and
Methods. Sequencing (Accession No. AF193553)showed that the
predicted DmUba2 contains 700 amino acids andhas 29% sequence
identity to yeast Uba2p and 48% identity tohuman hUba2, as observed
also by others (Bhaskar et al., 2000;Long and Griffith, 2000;
Donaghue et al., 2000). Like itshomologues and other E1-type
enzymes, DmUba2 contains anATP-binding motif (amino acids 26-31)
and the consensus Cys(C175) corresponding to those essential for
thiolester bondformation in other E1-type enzymes (Desterro et al.,
1999;Dohmen et al., 1995). Our original two-hybrid clone of
Dmubc9appeared to be full length by comparison to yeast UBC9and
oursequence for Dmubc9agreed with that reported by Joanisse etal.
(Joanisse et al., 1998).
We then raised antibodies to DmUba2 (see Materials andMethods).
The affinity-purified antibodies recognized mainlyone polypeptide
of apparent molecular weight ~97 kDa (Fig.1A), which is presumably
DmUba2 (predicted molecular weight,77.5 kDa). Support for this
conclusion was obtained byexpressing Dmuba2under GAL-promoter
control in yeast. Whencells were grown under inducing conditions,
the antibodiesrecognized primarily a polypeptide of apparent
molecular weight~97 kDa (Fig. 1B, lane 1) that was absent when
cells were grown
under repressing conditions (Fig. 1B, lane 2).
Similarlyanomalous low mobility on SDS-PAGE has been noted for
bothUba2p and hUba2 (Desterro et al., 1999; Dohmen et al.,
1995).
Identification of DmAos1 and its interaction with DmUba2In S.
cerevisiae, the E1 enzyme for ubiquitin activation is the1024
amino-acid Uba1p (McGrath et al., 1991). In contrast,Smt3p is
activated by a heterodimer of the 636 amino-acidUba2p, which is
related to the C-terminal part of Uba1p, and the347 amino-acid
Aos1p, which is related to the N-terminal partof Uba1p (Johnson et
al., 1997). Similarly, the 700 amino-acidDmUba2 is related in
sequence (~40% identity over the ~225amino acids of the three
similarity boxes defined for other Uba1-type and Uba2-type enzymes
(Johnson et al., 1997; Okuma etal., 1999)) to the C-terminal part
of the putative Drosophilaubiquitin-activating enzyme DmUba1 (EMBL#
Y15895).Therefore, we sought and identified a Drosophilahomologue
ofyeast AOS1among the ESTs from the Berkeley DrosophilaGenome
Project (BDGP) (see Materials and Methods).Sequencing (Accession
No. AF193554) showed that Dmaos1encodes a polypeptide of 337 amino
acids that has 28% sequenceidentity to yeast Aos1p and 40% identity
to the human Aos1phomologue Sua1 (Okuma et al., 1999), as also
observed by others(Bhaskar et al., 2000; Long and Griffith, 2000).
As expected,DmAos1 is related in sequence to the N-terminal part
ofDmUba1 (~37% identity over the ~202 amino acids of thesimilarity
boxes as defined previously (Johnson et al., 1997;Okuma et al.,
1999)), suggesting that a heterodimer of DmUba2and DmAos1 is the
DrosophilaSmt3/SUMO-activating enzyme.In support of this
hypothesis, we detected an interaction betweenfull-length DmUba2
and full-length DmAos1 in the two-hybridsystem (Table 3). An
attempt to use the two-hybrid system todelimit the region of DmUba2
responsible for its interaction withDmAos1 was unsuccessful (Table
3).
Identification of DmSmt3 and of DmSmt3-conjugatedproteinsWe next
sought and identified a homolog of SMT3/SUMO-1among the BDGP EST
clones (see Materials and Methods). Thepredicted DmSmt3 contains 90
amino acids with 48% identityto yeast Smt3p and 54% identity to
human SUMO-1, as alsoobserved by others (Bhaskar et al., 2000;
Huang et al., 1998;Lehembre et al., 2000). We generated polyclonal
antibodies (seeMaterials and Methods) and performed immunoblots on
flyextracts, expecting to detect both free DmSmt3 and DmSmt3-
Fig. 1. Immunoblot analyses using anti-DmUba2 and anti-DmSmt3
antibodies. (A) Extracts of wild-type embryos (seeMaterials and
Methods) were immunoblotted using affinity-purified anti-DmUba2
antibodies (lane 1) or the mock-affinity-purified fraction from
pre-immune serum (lane 2).(B) Yeast strain JD90-1A carrying
plasmids pIS2-ts10 andYCpIF2-DmUba2 (see Materials and Methods) was
grown inselective medium containing either 2% galactose (lane 1)
or2% glucose (lane 2) as the carbon source. Extracts
wereimmunoblotted using affinity-purified anti-DmUba2antibodies.
(C) Extracts of wild-type embryos were preparedeither with 20 mM
NEM (lane 1) or without NEM (lane 2)(see Materials and Methods) and
immunoblotted usingaffinity-purified anti-DmSmt3 antibodies.
Molecular weight markers are indicated. The arrow indicates the
free form of DmSmt3.
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1264
modified proteins. Because yeast and mammalian cells
containSmt3p/SUMO-1-specific isopeptidases (Gong et al., 2000;
Kimet al., 2000; Li and Hochstrasser, 1999; Li and
Hochstrasser,2000), which remove Smt3/SUMO from modified proteins,
weprepared extracts both with and without N-ethylmaleimide(NEM), an
isopeptidase inhibitor. In both extracts, the purifiedantibodies
recognized both a polypeptide of ~16 kDa(presumably free DmSmt3)
and many polypeptides of highermolecular weight (presumably
DmSmt3-conjugated proteins)(Fig. 1C). As expected, the higher
molecular-weight species were
both less abundant and of lower average molecular weight
whenextracts were prepared without NEM.
Interactions of DmUba2 and DmUbc9 with each otherand with
DmSmt3To test the hypothesis that DmUba2/DmAos1 and DmUbc9
areactivating and conjugating enzymes for DmSmt3, but not
forubiquitin (DmUb), we used in vitro protein-binding assays
toinvestigate the interactions among these proteins.
Becauseubiquitin and ubiquitin-like proteins undergo
proteolyticcleavage of their C-termini to leave the sequence
Gly-Gly, whichis essential for both activation and conjugation
(Kamitani et al.,1997; Melchior, 2000), we cloned DmSmt3 and DmUb
such thatthey terminated with Gly88 (DmSmt3) or Gly76 (DmUb)
andwere tagged with His6 at their N-terminal ends (see Materialsand
Methods). We then incubated purified His6DmSmt3 andpurified
His6DmUb with fly extracts, isolated the His6-taggedproteins using
Ni-NTA beads, and analyzed the associatedproteins. As expected, we
found that both DmUba2 and DmUbc9associated only with His6DmSmt3
and not with His6DmUb (Fig.2A,C). The anti-DmUba2 antibodies
detected not only the freeform of DmUba2 (~97 kDa) but also species
whose lowermobilities (Fig. 2A) suggested that they might
representDmUba2 conjugated to one, two, or three molecules of
DmSmt3(and/or some other ubiquitin-like molecule). To ask if
theinteractions of DmUba2 and DmUbc9 with DmSmt3 involvedthiolester
bonds, we repeated the experiments but omitted β-mercaptoethanol
(which reduces thiolester bonds) during samplepreparation. As
expected, the most abundant species now
Journal of Cell Science 115 (6)
Fig. 2.Conjugation of DmSmt3 to DmUba2 andDmUbc9 in vitro. Fly
embryo extractssupplemented with ATP and Mg2+ wereincubated with
His6DmSmt3, His6DmUb, or notagged protein, and proteins were
isolated usingNi-NTA beads and analyzed by SDS-PAGE
andimmunoblotting (see Materials and Methods).(A,B) Immunoblotting
using DmUba2-specificantibodies. SDS-PAGE was conducted
underreducing (A) or nonreducing (B) conditions. In(A), a sample of
unpurified lysate was alsoanalyzed. The species migrating at ~55
kDa ispresumably a breakdown product of DmUba2(see also Fig. 1A,B).
(C,D) Immunoblottingusing DmUbc9-specific antibodies. SDS-PAGEwas
conducted under reducing (C) ornonreducing (D) conditions. In C, a
sample ofunpurified lysate was also analyzed. In additionto DmUbc9
(predicted molecular weight, ~18kDa) (Joanisse et al., 1998),
anti-DmUbc9antibodies also recognize a polypeptide of ~25kDa; as
this species is evident both in the lysateand among proteins
isolated with His6DmUb, itmay be a DmUb-conjugating enzyme that
isrelated to DmUbc9. Molecular weight markersare shown for each
panel.
Table 3. Two-hybrid analysis of interaction betweenDmUba2 and
DmAos1a
Fusion plasmid
Fusion plasmidb DBD-DmAos1 AD-DmAos1
DmUba2-F 908 143c
DmUba2-N 52 15DmUba2-C 57 35Anillin 40 27No fusion 43 25
aTwo-hybrid tests between the indicated pairs of plasmids were
performedas described in Materials and Methods. Each entry shows
the units of β-galactosidase activity.
bThe DBD or AD fusion, as appropriate. DmUba2-F plasmids contain
full-length DmUba2; DmUba2-N and DmUba2-C plasmids contain the
N-terminus (amino acids 1-350) and C-terminus (amino acids 351-700)
ofDmUba2, respectively (see Materials and Methods).
cThe empty vector pJG4-5PL and the full-length AD-anillin clone
(seeMaterials and Methods) were used as negative controls with
DBD-DmUba2-F; each gave ~40 units of β-galactosidase activity.
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1265Smt3 conjugation system in Drosophila
observed with the anti-DmUba2 antibodies had an
apparentmolecular weight of ~116 kDa (Fig. 2B), consistent with
itsbeing DmUba2 with a single His6DmSmt3 linked by a
thiolesterbond. Similarly, the anti-DmUbc9 antibodies now revealed
anadditional species with an apparent molecular weight of ~30
kDa(Fig. 2D), presumably representing DmUbc9 with a
singleHis6DmSmt3 linked by a thiolester bond.
We also used immunoprecipitation to ask whether DmUba2and DmUbc9
interact in vivo. When immunoprecipitates wereprepared from embryo
extracts using antibodies to either protein,the other protein was
detected by immunoblotting (Fig. 3). Takentogether, the results of
in vitro binding assays andcoimmunoprecipitation suggest that
DmUba2/DmAos1 andDmUbc9 are indeed the activating and conjugating
enzymes,respectively, for DmSmt3 and that they may form a
complexcontaining both the E1-type and E2-type enzymes.
Localization of DmUba2 during embryogenesisTo begin
investigating the roles of the DmSmt3-conjugationpathway in
Drosophila, we used immunofluorescence andconfocal microscopy to
characterize the intracellular localizationof DmUba2. Yeast Uba2p
is concentrated in the nucleus(Dohmen et al., 1995), but the
localization of the homologousenzyme has not been examined in
multicellular organisms.Interestingly, we observed that DmUba2 is
not exclusivelynuclear during early embryogenesis. Before migration
of thenuclei to the embryo cortex after nuclear division 9,
DmUba2was found largely in the cortex, and its distribution
thereappeared homogenous (data not shown). During the
interphasepreceding nuclear division 10, DmUba2 gradually
becameorganized into a cap corresponding approximately to the
corticalactin cap that forms over each nucleus (Fig. 4, A1-A3,
C1-C3).DmUba2 was also found in the deeper cytoplasm (Fig. 4,
B1-B3, C1-C3) and gradually moved into the nuclei (Fig. 4,
B2-B3,C2-C3). During mitosis, DmUba2 was dispersed in the cortexand
in the cytoplasm near the cortex (Fig. 4, A4, B4, C4). Duringthe
three subsequent nuclear cycles, the cap-like localization(Fig. 4,
D1-D3, F1-F3, G1-G2, I1-I2), progressive nuclearaccumulation (Fig.
4, E1-E3, F1-F3, H1-H2, I1-I2), anddispersion during mitosis (Fig.
4, D4, E4, F4, G3, H3, I3) ofDmUba2 remained evident. However,
during the successivecycles, the cap-like cortical localization
became more organizedand the degree of nuclear enrichment became
more pronounced.By cycle 13, although some DmUba2 still localized
to the cortexduring interphase, it was predominantly nuclear (Fig.
4, G1-G2,H1-H2, and I1-I2).
Because DmUba2 was identified by its two-hybrid interactionwith
Sep1 and Sep2, we examined carefully whether DmUba2colocalized with
the septins either at the cellularization front orin cleavage
furrows in mitotic domains after gastrulation.
Duringcellularization, most DmUba2 localized to nuclei,
accumulatingpreferentially at their apical ends (Fig. 5A,C). We did
not detectDmUba2 at the cellularization front. However, some
DmUba2remained at the cortex, where diffuse septin staining was
alsoobserved (Fig. 5A,C). During mitosis in older embryos,DmUba2
spread throughout the cell but did not becomedetectably
concentrated in cleavage furrows (Fig. 5E-G, cells 1and 2 and
inset); it then moved back into the nuclei after mitosis,with no
detectable concentration at the midbody (Fig. 5E-G,cells 3 and 4).
Thus, we detected no substantial colocalizationof DmUba2 and
septins in early embryogenesis. However, itremains possible that
DmUba2 could interact with septins at thecortex in
syncytial-blastoderm embryos, during cellularization,or in mitotic
cells. DmUba2 was concentrated in nuclei ofnondividing cells and
dispersed throughout the cell duringmitosis throughout embryonic
stages 6 to 15 (Fig. 4J,K) (datanot shown). This was particularly
striking in the CNS, where theseptins, in contrast, are enriched in
axons (Fares et al., 1995;Neufeld and Rubin, 1994) (J. C. Adam et
al., unpublished) (Fig.6D).
We also examined DmUba2 localization during oogenesis.DmUba2
localized to the nuclei of both germ cells and somaticfollicle
cells in the germarium (Fig. 6A, green arrowhead; Fig.6C). After
encapsulation of the germ-line cells by the folliclecells, DmUba2
remained localized to follicle cell nuclei (Fig.6A, white arrow;
Fig. 6B). DmUba2 localization to nurse cellsdecreased as egg
chamber development progressed (Fig. 6A,B,green arrows), but it
remained enriched in the oocyte nucleus(Fig. 6A, white arrowhead).
In contrast, Pnut localizes primarilyto the basal surface of the
follicle cells and is excluded fromnuclei (Fig. 6A).
Localization of DmSmt3 in embryos and cultured cellsThe
two-hybrid interactions between the septins and DmUba2and DmUbc9
might reflect a physiologically significant buttransient
interaction, such as might occur if Drosophilaseptins,like yeast
septins (Johnson and Blobel, 1999; Takahashi et al.,1999), are Smt3
modified. To explore this possibility, we usedimmunofluorescence to
examine DmSmt3 localization incultured cells and in cellularizing
and older embryos. DmSmt3did not colocalize detectably with the
septins at thecellularization front (Fig. 5B,D). Instead, DmSmt3
localized to
Fig. 3.Coimmunoprecipitation of DmUba2 and
DmUbc9.Immunoprecipitates were prepared from embryo lysatesusing
the antibodies indicated (see Materials and Methods)and analyzed by
immunoblotting using (A) DmUba2-specific antibodies or (B)
DmUbc9-specific antibodies. Theimmunoprecipitation using anti-myc
antibodies serves as anegative control. The dark band at ≥20 kDa in
(B) ispresumably an allotypic form of rabbit IgG light chain in
theanti-DmUbc9 antibodies that is recognized by the HRP-conjugated
goat anti-rabbit-IgG.
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1266
nuclei, with a particular enrichment at their apical ends, as
didDmUba2 (Fig. 5A-D). In cultured cells and in cells of
post-gastrulation embryos, DmSmt3 was concentrated in
nucleithroughout interphase (Fig. 5H, cells 1 and 2; Fig. 5K-M,
cells1-3). During mitosis, DmSmt3 initially appeared to
spreadthroughout the cell (Fig. 5K-M, cell 5). However,
duringmetaphase, DmSmt3 appeared to concentrate in the region of
thechromosomes (Fig. 5I; Fig. 5K-M, cell 4); this was confirmed
by localizing DmSmt3 relative to the mitotic spindle (Fig.
5J).Strikingly, DmSmt3 was also found concentrated in a spot
atcleavage furrows and midbodies both in cultured cells (Fig.
5H,cells 3 and 4) and in dividing embryonic cells (Fig. 5K-M,
cells6-8, 10 and 13); this spot overlapped, but did not appear
tocoincide fully, with the concentration of the septins in
thesefurrows. DmSmt3 was not enriched at the cleavage furrows
earlyin furrow formation (Fig. 5K-M, cells 9, 11 and 12), but
only
during later stages, and it remained concentrated in themidbody
after most DmSmt3, and essentially all of theDmUba2, had
reaccumulated in nuclei (Fig. 5H, cell 4;K-M, cells 6-8, 10, and
13; and compare with E-G,cells 3 and 4).
We also examined DmSmt3 localization duringother stages of
embryogenesis. During the syncytialcell cycles, DmSmt3 was
concentrated in the nucleiduring interphase (Fig. 6G) and appeared
to localize tothe chromosomes during mitosis (Fig. 6F). LikeDmUba2,
DmSmt3 localized primarily to the nuclei ofnon-mitotic cells
throughout the rest of embryogenesis(Fig. 6H), including in the CNS
(where the septins, incontrast, localized to axons) (Fig. 6E).
The concentration of DmSmt3 in late cleavagefurrows and
midbodies suggested that one or moreof the Drosophila septins might
be modified byDmSmt3. We thus tried various experimentalapproaches
to look for DmSmt3-modified septins.First, we used immunoblotting
to look for higher-molecular-weight forms of Pnut, Sep1 or
Sep2,which might represent Smt3-modified proteins, inextracts from
embryos, adults, and cultured Clone-8and S2 cells. Second, we
immunoprecipitated Pnut,Sep1, and Sep2 from embryonic extracts
andanalyzed the precipitates by immunoblotting usinganti-DmSmt3
antibodies. Third, we preparedimmunoprecipitates from embryonic
extracts usinganti-DmSmt3 antibodies and analyzed theprecipitates
by immunoblotting using anti-Pnut,anti-Sep1, and anti-Sep2
antibodies. In each case,we did the experiments both in the
presence and the
Journal of Cell Science 115 (6)
Fig. 4. Localization of DmUba2 during embryogenesis.(A-I)
Localization of DmUba2 in syncytial-blastodermembryos during
nuclear cycle 10 (A-C), 11 (D-F), and 13(G-I). Surface views of
embryos in interphase (A1-A3, D1-D3, G1, and G2) or mitosis (A4,
D4, and G3) are shown;optical sections parallel to the surface and
through themiddle of nuclei in interphase (B1-B3, E1-E3, H1, and
H2)or mitosis (B4, E4, and H3) and medial longitudinaloptical
sections of embryos in interphase (C1-C3, F1-F3,I1, and I2) or
mitosis (C4, F4, and I3) are also shown. Eachvertical set of three
images (e.g., A1, B1, and C1) showsthe same embryo. Each row of
interphase images (e.g., A1,A2, and A3) is ordered according to the
presumedsequence of the embryos within interphase, with theearliest
point on the left (e.g., A1), based on theassumption that DmUba2
must gradually reaccumulate inthe nucleus after its dispersion
during mitosis.(J,K) Localization of DmUba2 in older embryos in
stage 9(J) or 12 (K). Medial longitudinal optical sections
areshown. Arrow indicates a mitotic domain. Bar, 10 µm(A-I); 50 µm
(J and K).
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1267Smt3 conjugation system in Drosophila
absence of the isopeptidase inhibitorNEM. None of these
approachesdetected DmSmt3 modification of Pnut,Sep1, or Sep2 (data
not shown). Thesenegative results may mean that theseptins are not
Smt3 modified. However,our immunofluorescence studiesdetected
colocalization of Smt3 with theseptins only for a brief period at
the endof mitosis, and even at this stage theoverlap was not
complete. Thus even ifseptins are Smt3 modified during thisperiod,
they would probably compriseonly a very small fraction of the
totalseptins in the embryo and thus mighthave escaped our
detection.
DiscussionPossible interaction of the septins andthe Smt3
conjugation systemThe Drosophila septins appear to beessential for
cytokinesis in at least somecell types, and it is likely that they
have avariety of non-cytokinesis roles as well(see Introduction).
Because studies inyeast suggest that a primary function ofthe
septins is to serve as a matrix ortemplate for the organization of
otherproteins at the cell surface, theidentification of
septin-interacting proteins should be critical tothe elucidation of
septin function in Drosophila. This studybegan with an attempt to
identify such proteins using the yeasttwo-hybrid system. Of 27
positive clones identified with threeseptin baits, 17 contained
fragments of the septin genesthemselves. Because other evidence
suggests strongly that theseptins interact with each other in vivo
(Beites et al., 1999; DeVirgilio et al., 1996; Fares et al., 1995;
Field et al., 1996; Frazieret al., 1998; Hsu et al., 1998; Longtine
et al., 1996), this resultsuggests that the baits used were good
and that the screen wasotherwise of high fidelity. Thus, it seems
likely that at least some
of the other positive clones represent genes whose
productsreally interact with septins in vivo.
Of the six non-septin genes identified in the screens, onlytwo
have been investigated in detail as yet. These genes,Dmuba2and
Dmubc9, encode the Drosophilahomologues ofyeast Uba2p and Ubc9p,
which catalyze the activation andconjugation, respectively, of the
ubiquitin-like moleculeSmt3p (see Introduction). Several lines of
evidence suggestthat the two-hybrid interactions observed between
DmUba2,DmUbc9, and the septins reflect physiologically
significantinteractions. First, among the 10 positive clones that
did not
Fig. 5. Localization of DmUba2 and DmSmt3during cellularization,
in mitotic domains, andin cultured cells. Some cells are numbered
forreference in the text. (A-D) Embryos early(A,B) or late (C,D) in
cellularization werestained for Pnut (red) and either DmUba2(green
in A,C) or DmSmt3 (green in B,D).(E-G, I-M) Mitotic domains in
extended-germband embryos were double stained forDmUba2 (E, E
inset, and green in G and Ginset) or DmSmt3 (K and green in I,J,M)
andeither Pnut (F,L, and red in G,I,M) or tubulin(F inset and red
in G inset and J). Arrowheadsin I,J indicate concentrations of Smt3
in theregions of the chromosomes. Images of threedifferent embryos
are shown in K-M. (H)Clone-8 cells undergoing cytokinesis
werestained for DmSmt3 (green) and Pnut (red).Bars, 10 µm (A-G,
same magnification; K-Msame magnification).
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1268
encode septins, four contained either Dmuba2or Dmubc9,and Dmuba2
fragments were isolated with two differentseptin baits. Second,
because DmUba2 and DmUbc9 alsointeract with each other (see
Results), the identification ofboth genes independently in screens
using septin baitssuggests that the interactions are relevant.
Third, while ourstudies were in progress, it became clear that
yeast septinsare extensively modified by Smt3p, although the
functionalsignificance of that remains uncertain (Johnson and
Blobel,1999; Takahashi et al., 1999; Takahashi et al., 2001;
Johnsonand Gupta, 2001) (P. Meluh, personal communication).Fourth,
several other groups also isolated Uba2 and/or Ubc9in two-hybrid
screens using other protein baits. Several ofthese interactors,
including Drosophila calcium/calmodulin-dependent kinase II
(CAM-kinase II; Long and Griffith,2000) and Dorsal (Bhaskar et al.,
2000), were subsequentlyshown to be Smt3 modified.
Other genetic data may also reflect an interaction between
theseptins and the DmSmt3 system. The pnut septin mutation
wasoriginally identified as an enhancer of a sinamutation that
affectsR7 photoreceptor development (Carthew et al., 1994;
Neufeldand Rubin, 1994). Although the significance of this
geneticinteraction remains unclear, it may reflect the
recentlydiscovered crosstalk between Smt3/SUMO modification
andubiquitination. Mammalian SUMO-1 is conjugated to theprotein
Mdm2, a RING-finger E3 ubiquitin ligase involved inp53 degradation
(Buschmann et al., 2000). Modification by
SUMO-1 appears to regulate Mdm2 activity and hence the levelof
p53, probably by regulating the ubiquitination anddegradation of
Mdm2 itself. Other RING-finger proteins,including DrosophilaSina,
interact with Ubc9 family proteinsand/or are modified by Smt3/SUMO
(Duprez et al., 1999; Hu etal., 1997). Thus, it seems possible that
DmSmt3 modificationregulates the activities of Sina, such as its
role in downregulatingthe transcriptional repressor Tramtrack (one
of whose isoformsis itself DmSmt3 modified) (Lehembre et al., 2000;
Li et al.,1997; Neufeld et al., 1998; Tang et al., 1997), and that
Pnut playsa role in mediating the requisite interactions.
Finally, in several types of dividing Drosophila cells, wefound
that DmSmt3 colocalizes with septins in the cleavagefurrows and/or
midbodies during cytokinesis. Interestingly,DmSmt3 is not enriched
in the furrow during the early stagesof furrow formation but only
later, at a time when mostDmUba2 has moved back into the nuclei.
Although we wereunable to detect DmSmt3 modification of Sep1, Sep2,
or Pnut(despite considerable effort), it remains possible that one
ormore of these proteins is modified at low levels or that DmSmt3is
conjugated to Sep4 or Sep5. However, it is also possible thatthe
DmSmt3 in the midzone is conjugated to other proteins,such as the
‘chromosomal passenger proteins’ (Adams et al.,2001), which are
associated with the chromosomes and thenrelocate to the spindle
midzone in mitosis. The involvement ofsuch proteins in chromosome
segregation as well as incytokinesis might help to explain the
observations suggesting
Journal of Cell Science 115 (6)
Fig. 6. Localization of DmUba2 and DmSmt3 at other developmental
stages and in other cell types. (A-C) Localization of DmUba2 to
nucleiduring oogenesis. Egg chambers were double labeled with
anti-DmUba2 and anti-Pnut. Arrows and arrowheads indicate
structures discussed inthe text. (A) Ovariole with the germarium on
the left and successively older egg chambers moving left to right.
DmUba2 (top panel; green inbottom panel) and Pnut (middle panel;
red in bottom panel) are shown. (B) Overexposure of DmUba2 staining
of an ovariole similar to thatshown in A. (C) Higher magnification
of a germarium, showing enrichment of DmUba2 (green) in nuclei of
both follicle and germ cells, aswell as in the nuclei of the muscle
cells in the sheath surrounding the germarium. (D,E) Embryonic
central nervous system after double stainingfor Pnut (red) and
either DmUba2 (green in D) or DmSmt3 (green in E). (F,G) DmSmt3
localization to nuclei of syncytial blastoderm embryosduring
interphase (G) and to the chromosomes during mitosis (F). (H)
Localization of DmSmt3 (green) to interphase nuclei
duringembryogenesis. Embryos at stages 8 (H1), 10 (H2), and 14 (H3)
are shown.
-
1269Smt3 conjugation system in Drosophila
that the Smt3/SUMO system is involved in chromosomesegregation
(Apionishev et al., 2001; Meluh and Koshland,1995; Tanaka et al.,
1999).
It also remains unclear whether the Drosophilaseptins everserve
as a matrix/template for the localization of the DmSmt3conjugation
system. Although our immunofluorescence studiesshow that DmUba2 and
the septins are sometimes in the samecompartment, so that
interaction would be possible, we observedno persuasive
colocalization of the proteins. Thus, elucidation ofthe possible
interactions between the septins and the Smt3system in Drosophila,
and of their functional significance, willneed to await further
studies using other approaches.
Conservation and possible functions of the Smt3/SUMOconjugation
pathway in DrosophilaDespite these residual uncertainties, in the
course of our studieswe generated other valuable information about
the Smt3/SUMOconjugation system in Drosophila that complement and
extendrecent work by others on this system. For example,
ourbiochemical and two-hybrid studies indicate that there
aremultiple DmSmt3-modified proteins, that
DmSmt3-specificisopeptidases probably exist, that DmUba2 and
DmAos1interact with each other, and that both DmUba2 and
DmUbc9become conjugated to DmSmt3, but not to DmUbiquitin,
bythiolester bonds. While our studies were in progress,
relatedfindings were also made by other investigators who were led
byother routes to the Smt3/SUMO system in Drosophila. Inparticular,
several other proteins were shown to interact withDmUba2 and DmUbc9
using the two-hybrid system, andmultiple DmSmt3-modified proteins
were observed (Bhaskar etal., 2000; Donaghue et al., 2001; Joanisse
et al., 1998;Lehembre et al., 2000; Long and Griffith, 2000; Ohsako
andTakamatsu, 1999), supporting the hypothesis that DmSmt3 isindeed
conjugated to a variety of proteins in vivo. In addition,the
DmUba2/DmAos1 interaction and the conjugation ofDmSmt3 to DmUba2
and DmUbc9 by thiolester bonds werealso observed using other
methods (Lehembre et al., 2000; Longand Griffith, 2000). Finally,
it was shown that Dmubc9canfunctionally complement a yeast
ubc9mutation (Joanisse et al.,1998; Ohsako and Takamatsu, 1999).
Taken together, theseresults make clear that the Smt3/SUMO
conjugation system isclosely conserved between Drosophila, yeast,
and mammals.Our data also show that DmUba2 and DmUbc9 form a
complexin vivo, suggesting that the conjugation machinery may act
ina concerted fashion.
The studies in other laboratories also provided clues topossible
functions of modification by DmSmt3. In particular, thesemushi(Epps
and Tanda, 1998) and lesswright(Apionishev etal., 2001) mutations
are both in Dmubc9, suggesting (from theirmutant phenotypes) that
DmUbc9 has roles in the nuclear importof the transcription factor
Bicoid and in meiotic chromosomesegregation. In addition, two other
transcriptional regulators,Dorsal and Tramtrack, as well as
CAM-kinase II, have also beenshown to be modified by DmSmt3
(Bhaskar et al., 2000;Lehembre et al., 2000; Long and Griffith,
2000). In the case ofDorsal, as with Bicoid, DmSmt3 conjugation
appears to promotenuclear localization, whereas Tramtrack
modification may helpto regulate its activity and/or its
degradation by the proteasome,as discussed above. The modification
of CAM-kinase II mayregulate its activity.
Although most suggested functions of the Smt3/SUMOsystem in
Drosophila, as well as in yeast and mammalian cells,center on
nuclear proteins, our immunofluorescence and two-hybrid data
support the hypothesis that there are alsocytoplasmic targets.
First, as discussed above, it remains likelythat in Drosophila, as
in yeast, there is an interaction betweenthe Smt3/SUMO system and
the septins, which appear to beexclusively cytoplasmic proteins.
Second, some DmSmt3-modified proteins appear to remain both at the
embryo cortexduring cellularization and in the midbodies that
remain after thenuclear envelopes have reformed at the end of
cytokinesis. Third,although DmUba2 (this study) and DmUbc9
(Joanisse et al.,1998; Lehembre et al., 2000) are found primarily
in nuclei,considerable DmUba2 is also found in the cytoplasm and at
thecortex during the syncytial-blastoderm stage, suggesting
thatDmSmt3 modification of cortical and/or cytoplasmic
proteinscould occur. Finally, the cell-cycle-regulated
translocation ofDmUba2 between cytoplasm and nucleus both in
syncytial-blastoderm and in post-cellularization embryos may
suggest thatthe partitioning of the DmSmt3-conjugation system
between thecytoplasm and the nucleus is important and thus well
regulatedduring embryonic development.
We thank Erica Johnson and Pam Meluh for stimulating
discussions;Robert Tanguay for antibodies; Julie Brill for
constructs; Tony Perdue andSusan Whitfield for help with confocal
microscopy and photography; andmembers of the Pringle and Peifer
laboratories for encouragement andcomments on the manuscript. This
work was supported by NationalInstitute of Health grant GM 52606 to
J.R.P. and M.P. and by NIHPostdoctoral Fellowship GM19981 to
K.G.H.
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