smt3 conjugation system in drosophila · 2002. 2. 27. · characterization of the...

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

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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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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

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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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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


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.


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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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


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.


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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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


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).


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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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


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.


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.

ReferencesAdam, J. C., Pringle, J. R. and Peifer, M.(2000). Evidence for functional

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