In a two hybrid screen designed to identify proteins that interact with small heat shock proteins (sHsps), a Drosophila melanogaster homologue of yeast and human ubc9 (Dmubc9) was found to interact with Drosophila Hsp23. Further, two-hybrid system analysis reveals DmUbc9 interaction with Drosophila and mammalian Hsp27. In situ hybridization localizes Dmubc9 as a doublet at locus 21D on chromosome 2L, and genomic cloning of the gene reveals a single open reading frame without introns. The predicted Dmubc9 protein sequence shares a very high level of homology with mouse (85.4%) and human (> or = 82.9%) Ubc9. Genetic complementation analysis show that Dmubc9 functionally rescues a temperature-sensitive S. cerevisiae ubc9ts mutant. Co-immunoprecipitation with antibody raised against DmUbc9 confirms the interaction with Drosophila Hsp23 and Hsp26 and preferentially with Hsp27 (Joanisse, 1998).
The maternal transcript of the anterior segmentation gene bicoid (bcd) is localized at the anterior pole of the Drosophila egg and translated to form a gradient in the nuclei of the syncytial blastoderm embryo after fertilization. The nuclear gradient of Bcd protein (a transcription factor) leads to differential expression of zygotic segmentation genes. The rapid nuclear division in the early zygote requires that Bcd quickly enters the nuclei after each mitosis using an active nuclear import system. Nuclear transport depends on the asymmetrical distribution of two forms of the small GTPase Ran: Ran-GTP that is concentrated in the nucleus and Ran-GDP in the cytoplasm. Ran requires RanGTPase-activating protein-1 (RanGAP1) on the cytoplasmic side of nuclear pore complexes to convert Ran-GTP to Ran-GDP. In vitro studies with vertebrate proteins demonstrate that the RanGAP1 associated with the nuclear pore complex is modified with small ubiquitin related modifier-1 (SUMO-1) by a ubiquitin-conjugating enzyme (E2 enzyme). Mutation of the Drosophila semushi (semi) gene, which encodes an E2 enzyme, blocks nuclear import of Bcd during early embryogenesis and results in misregulation of the segmentation genes that are Bcd targets. Consequently, semi embryos have multiple defects in anterior segmentation. This study demonstrates that an E2 enzyme is required for nuclear transport during Drosophila embryogenesis. semi could be responsible for modification of other proteins essential for Bcd nuclear transport. Nevertheless, these results indicate the possible connection of the function of an E2 enzyme of the Ubc9 family to nuclear import in Drosophila. Hunchback is accumulated in the nucleus in a normal fashion in semi mutants. In semi mutants, posterior segmentation genes function correctly (Epps, 1998).
Drosophila Uba2 and Ubc9 SUMO-1 conjugation enzyme homologs (DmUba2 and DmUbc9) were isolated as calcium/calmodulin-dependent kinase II (CaMKII) interacting proteins by yeast two-hybrid screening of an adult head cDNA library. At least one isoform of Drosophila neuronal CaMKII is conjugated to DmSUMO-1 in vivo. The interactions observed in the two-hybrid screen may therefore reflect catalytic events. To understand the role of SUMO conjugation in the brain, a characterization of the system was undertaken. The other required components of the system, Drosophila Aos1 and SUMO-1 (DmAos1 and DmSUMO-1), were identified in expressed sequence tag data base searches. Purified recombinant DmUba2/DmAos1 dimer can activate DmSUMO-1 in vitro and transfer DmSUMO-1 to recombinant DmUbc9. DmSUMO-1 conjugation occurs in all developmental stages of Drosophila and in the adult central nervous system. Overexpression of a putative dominant negative DmUba2(C175S) mutant protein in the Drosophila central nervous system resulted in an increase in overall DmSUMO-1 conjugates and a base-sensitive p120 species, which is likely to be DmUba2(C175S) linked to endogenous DmSUMO-1 through an oxygen ester bond. Overexpression of DmUba2(wt) protein in vivo also led to increased levels of DmSUMO-1 conjugates. High level overexpression of either DmUba2(wt) or DmUba2(C175S) in the Drosophila central nervous system caused pupal and earlier stage lethality. Expression in the developing eye led to a rough eye phenotype with retinal degeneration. These results suggest that normal SUMO conjugation is essential in the differentiated nervous system and reveal a potential novel mechanism that regulates neuronal calcium/calmodulin-dependent protein kinase II function (Long, 2000).
To identify proteins that regulate the function of Dorsal, a yeast two-hybrid screen was used to search for genes encoding Dorsal-interacting proteins. Six genes have been identified, including two that encode previously known Dorsal-interacting proteins (Twist and Cactus); three that encode novel proteins, and one that encodes Drosophila Ubc9 (DmUbc9). The name 'Ubc9' reflects the homology of this protein to ubiquitin-conjugating enzymes. However, recent studies on yeast and human Ubc9 have shown that this enzyme primarily conjugates the yeast protein Smt3p or its human homologs SMT3A, SMT3B, and SMT3C rather than ubiquitin to proteins. DmUbc9 binds and conjugates Drosophila Smt3 (DmSmt3) to Dorsal. In cultured cells, DmUbc9 relieves inhibition of Dorsal nuclear uptake by Cactus, allowing Dorsal to enter the nucleus and activate transcription. The effect of DmUbc9 on Dorsal activity is potentiated by the overexpression of DmSmt3. A DmSmt3-activating enzyme, DmSAE1/DmSAE2, has been identified, and found to further potentiate Dorsal-mediated activation (Bhaskar, 2000).
Smt3 homologs have been cloned from eukaryotes as diverse as yeast, Arabidopsis, and humans. In general, these proteins display greater than 50% identity with one another but also roughly 20% identity with ubiquitin. The identification of the components of the Smt3 conjugation pathway in yeast, humans, and now Drosophila has revealed that Smt3 conjugation and ubiquitin conjugation proceed by similar pathways. Both pathways require an activating enzyme, or E1 protein, which becomes covalently attached to ubiquitin or Smt3 via a high energy thioester bond, and a conjugating enzyme, or E2 protein, which accepts ubiquitin or Smt3 from the E1 protein forming a second thioester-linked covalent complex. Ubiquitin or Smt3 is then transferred to an epsilon-amino group on a final protein substrate. The transfer of ubiquitin from the E2 protein to the final substrate often requires a ubiquitin ligase, or E3 protein. In contrast, an E3-type protein is apparently not required for Smt3 conjugation (Bhaskar, 2000 and references therein).
Although ubiquitin conjugation targets proteins for proteasomal degradation, Smt3 conjugation appears to serve other purposes. Originally identified in yeast as an enzyme required for proper cell cycle progression, Ubc9 has been found to physically interact with a diverse array of proteins, including RanGAP1, PML (promyelocytic leukemia protein), bleomycin hydrolase, E2A, androgen receptor, and c-Rel. Association of human Ubc9 with RanGAP1 results in the conjugation of RanGAP1 to the Smt3 homolog SMT3C/SUMO-1 (small ubiquitin-related modifier), allowing it to bind RanBP2 at the nuclear periphery. This allows RanGAP1 to stimulate GTP hydrolysis by Ran. Only SUMO-1-conjugated RanGAP1 binds to RanBP2, implying that SMT3C and Ubc9 are required for nuclear import. In the case of PML, interaction with Ubc9 and subsequent SUMO-1 conjugation is essential for targeting PML to discreet subnuclear structures known as PML-bodies or nuclear dots. In acute promyelocytic leukemia cells, the subnuclear localization of PML is altered, suggesting that improper SUMO-1 conjugation may trigger oncogenesis. These studies argue that one function of Smt3 conjugation is to regulate the subcellular localization of proteins (Bhaskar, 2000 and references therein).
Although Smt3 conjugation may play a role in regulating Dorsal activity, a number of reports have implicated Ubc9 in the modulation of transcriptional activation by other Rel family proteins. For example, SUMO-1-conjugated IkappaB is resistant to degradation and, accordingly, SUMO-1 and Ubc9 work together to inhibit activation of an NFkappaB-dependent reporter. This contrasts with the current findings, which show that the Smt3 conjugation pathway activates Dorsal-dependent reporters. This difference could relate to inherent differences between the NFkappaB/IkappaB and Dorsal/Cactus pathways. However, an earlier report suggests that mammalian Ubc9 can enhance Rel protein function via an interaction with NFkappaB and/or IkappaB. Thus, an alternative explanation for the different effects of Smt3 conjugation on Rel protein activity could be that different Smt3 family proteins have different functions. An alignment of DmSmt3 with the three members of the human SMT3 family reveals that DmSmt3 displays significantly higher homology to SMT3A and SMT3B (77% and 75%, respectively) than to SMT3C/SUMO-1 (55%). Thus, DmSmt3, SMT3A, and SMT3B appear to define an Smt3 subfamily that is distinct from SMT3C/SUMO-1. Perhaps SMT3C/SUMO-1 antagonizes transcriptional activation by Rel proteins, whereas SMT3A/B-like proteins (such as DmSmt3) enhance Rel protein function (Bhaskar, 2000 and references therein).
The Smt3 conjugation system may also function at other levels in the regulation of Rel family protein activity. For example, Ubc9 has been shown to associate with the type I TNFalpha receptor and MEKK1 and to synergize with MEKK1 to activate an NFkappaB-dependent reporter. Although no DmSmt3-Dorsal conjugate could be detected in cells that were simultaneously co-transfected with Dorsal, DmUbc9, and DmSmt3, the level of conjugation is low: no more than about 10% of the Dorsal protein is found in the DmSmt3-conjugated form. Perhaps the conjugation of DmSmt3 to Dorsal is transient. Perhaps Dorsal and DmSmt3 are deconjugated as soon as Dorsal enters the nucleus. In accord with this idea, recent observations suggest that a dynamic equilibrium may exist between Smt3-conjugated and unconjugated protein species. In yeast, the vast majority of cellular Smt3p is conjugated to other proteins, although the population of proteins that is covalently modified changes during the cell cycle. Furthermore, a yeast enzyme capable of catalyzing the deconjugation reaction has been identified, and homologs of this enzyme appear to exist in many other eukaryotic species (Bhaskar, 2000 and references therein).
A genetically defined locus, termed semushi (Epps, 1998) is identical with DmUbc9. Experiments employing the semushi allele suggest that DmUbc9 may be necessary for the nuclear import of the anteroposterior patterning morphogen Bicoid. Embryos lacking maternally supplied DmUbc9 have multiple patterning defects of varying penetrance. Because of the complex nature of these defects, their characterization will require extensive phenotypic analysis and the generation of additional DmUbc9 alleles. The possibility that DmUbc9 has pleiotropic developmental roles is not surprising given increasing evidence for wide spread roles of Smt3 conjugation in transcription factor function and in the targeting of proteins to their proper subcellular locales (Bhaskar, 2000 and references therein).
A variety of transcription factors are targets for conjugation to the ubiquitin-like protein Smt3 (also called SUMO). While many such factors exhibit enhanced activity under conditions that favor conjugation, the mechanisms behind this enhancement are largely unknown. The Drosophila rel family factor Dorsal is a substrate for Smt3 conjugation. The conjugation machinery enhances Dorsal activity at least in part by counteracting the Cactus-mediated inhibition of Dorsal nuclear localization. Smt3 conjugation occurs at a single site in Dorsal (lysine 382), requires just the Smt3-activating and -conjugating enzymes, and is reversed by the deconjugating enzyme Ulp1. Mutagenesis of the acceptor lysine eliminates the response of Dorsal to the conjugation machinery and results in enhanced levels of synergistic transcriptional activation. Thus, in addition to controlling Dorsal localization, Smt3 also appears to regulate Dorsal-mediated activation, perhaps by modulating an interaction with a negatively acting nuclear factor. Finally, since Dorsal contributes to innate immunity, the role of Smt3 conjugation in the immune response was investigated. The conjugation machinery is required for lipopolysaccharide-induced expression of antimicrobial peptides in cultured cells and larvae, suggesting that Smt3 regulates Dorsal function in vivo (Bhaskar, 2002).
The ubiquitin-related SUMO-1 modifier can be covalently attached to a variety of proteins. To date, four substrates have been characterized in mammalian cells: RanGAP1, IkappaBalpha, and the two nuclear body-associated PML and Sp100 proteins. SUMO-1 modification has been shown to be involved in protein localization and/or stabilization and to require the activity of specialized E1-activating and E2 Ubc9-conjugating enzymes. SUMO-1 homologs have been identified in various species and belong to the so-called Smt3 family of proteins. The Drosophila homologs of mammalian SUMO-1 and Ubc9 (termed dSmt3 and dUbc9, respectively) have been characterized. dUbc9 is the conjugating enzyme for dSmt3 and dSmt3 can covalently modify a number of proteins in Drosophila cells in addition to the human PML substrate. The dSmt3 transcript and protein are maternally deposited in embryos, where the protein accumulates predominantly in nuclei. Similar to its human counterpart, dSmt3 protein is observed in a punctate nuclear pattern. Tramtrack 69 (Ttk69), a repressor of neuronal differentiation, is a bona fide in vivo substrate for dSmt3 conjugation. Both the modified and unmodified forms of Ttk69 can bind to a Ttk69 binding site in vitro. Moreover, dSmt3 and Ttk69 proteins colocalize on polytene chromosomes, indicating that the dSmt3-conjugated Ttk69 species can bind at sites of Ttk69 action in vivo. Altogether, these data indicate a high conservation of the Smt3 conjugation pathway and further suggest that this mechanism may play a role in the transcriptional regulation of cell differentiation in Drosophila flies (Lehembre, 2000).
The identification of the transcriptional repressor Ttk69 as a substrate of the dSmt3 conjugation pathway suggests that this mode of posttranslational modification may play a direct role in the modulation of transcriptional regulation. Supporting this possibility, the localization of dSmt3 at particular chromosomal sites shows that the dSmt3 modification can be chromosome associated. Its partial colocalization with Ttk69 and the ability of the dSmt3-modified Ttk69 protein to bind Ttk69 sites are also consistent with the binding of modified Ttk69 to a subset of Ttk69 recognition elements. Although Ttk69 is the first transcription factor shown to be modified by the SUMO-1/Smt3 homologs, it seems likely that SUMO-1 also modifies several transcription factors in mammalian cells, as suggested by the observed interaction in a two-hybrid assay of Ubc9 with E1A, IB, WT1, Jun, p53, ATF2, ETS-1, the glucocorticoid receptor, and other nuclear proteins and thus may perform a more general role in transcriptional regulation. These data also indicate that the pattern of covalent modification of Ttk69 may be more complex. In particular, Ttk69 can be phosphorylated as well as conjugated with dSmt3. Notably, general inhibition of serine/threonine phosphorylation prevents dSmt3 conjugation, although it is uncertain whether this is a consequence of a reduction in substrate availability or conjugating activity (Lehembre, 2000 and references therein).
The biological role and consequences of the conjugation of dSmt3 to Ttk69 are unclear. Among several possibilities would be effects on the targeting of the repressor to specific chromosomal sites or on its interaction with specific protein partners. Another attractive hypothesis is that dSmt3 modification might antagonize the degradation of Ttk69 by a proteasome-dependent pathway. Indeed, it has recently been suggested that in human cells, SUMO-1 modification of IB might serve to block signal-induced ubiquitination and thus degradation of IB. In this context it is intriguing that Sina interacts directly with and destabilizes the other isoform of Ttk, Ttk88, but that no comparable interaction of Sina and Ttk69 was observed in a two-hybrid assay. Nevertheless, Ttk69 levels are stabilized in SL2 cells by MG132, an inhibitor of proteasome-mediated proteolysis. It is therefore suggested that dSmt3 modification might provide a mechanism for the differential stabilization of splicing isoforms, such as Ttk69 and Ttk88, that are transcribed from the same promoter. Genetic analysis of dSmt3 mutants in Drosophila should hopefully lead to a better understanding of the role of dSmt3 modification in the transcriptional regulation of sense organ development (Lehembre, 2000).
The septins are a family of proteins involved in cytokinesis and other aspects of cell-cortex organization. In a two-hybrid screen designed to identify septin-interacting proteins in Drosophila, several genes were isolated, including homologues (Dmuba2 and Dmubc9) of yeast UBA2 and UBC9. Yeast Uba2p and Ubc9p are involved in the activation and conjugation, respectively, of the ubiquitin-like protein Smt3p/SUMO, which becomes conjugated to a variety of proteins through this pathway. Uba2p functions together with a second protein, Aos1p. The Drosophila homologues of AOS1 (Dmaos1) and SMT3 (Dmsmt3) were also cloned and characterized. Biochemical data suggest that DmUba2/DmAos1 and DmUbc9 indeed act as activating and conjugating enzymes for DmSmt3, implying that this protein-conjugation pathway is well conserved in Drosophila. Immunofluorescence studies showed that DmUba2 shuttles between the embryonic cortex and nuclei during the syncytial blastoderm stage. In older embryos, DmUba2 and DmSmt3 are both concentrated in the nuclei during interphase but dispersed throughout the cells during mitosis, with DmSmt3 also enriched on the chromosomes during mitosis. These data suggest that DmSmt3 could modify target proteins both inside and outside the nuclei. No concentration was observed of DmUba2 at sites where the septins are concentrated, and no DmSmt3 modification was detected of the three Drosophila septins tested. However, DmSmt3 localization to the midbody during cytokinesis was observed both in tissue-culture cells and in embryonic mitotic domains, suggesting that DmSmt3 modification of septins and/or other midzone proteins occurs during cytokinesis in Drosophila (Shih, 2002).
To identify proteins interacting with the Drosophila septins, two-hybrid screens were conducted using Pnut, Sep1, and Sep2 as baits. These screens identified 27 positive clones that proved to represent eight genes. Among these were the other septins, as expected from other data indicating that septins interact with one another. In addition, the screens identified Drosophila homologues (Dmuba2 and Dmubc9) of yeast UBA2 and UBC9, whose products are involved in the activation and conjugation of the ubiquitin-like molecule Smt3p/SUMO. These screening results and the recent discovery of Smt3p modification of septins in S. cerevisiae (Johnson, 1999; Takahashi, 1999) stimulated a study the Smt3p/SUMO conjugation machinery in Drosophila (Shih, 2002).
In further two-hybrid analyses, the C-terminal portion of DmUba2 interacted strongly with full-length Sep1 and Sep2 and with the N-terminal portion of Sep1. Interactions were also detected with the N-terminal portions of Pnut and Sep2 and with the C-terminal portions of Sep1 and Sep2. In contrast, a full-length AD-DmUba2 fusion showed none of these interactions, although other studies indicated that this fusion was functional for other interactions. Interestingly, full-length AD-DmUbc9 showed a pattern of interactions very similar to those seen with the C-terminal portion of DmUba2 (Shih, 2002).
The other genes identified in the screens had not been described previously; they were designated sip1-sip4 (for septin-interacting protein). sip1 (Accession No. AF221101; Drosophila genome annotation No. CG7238) encodes a protein with predicted P-loop and coiled-coil domains; it appeared to interact specifically with the C-terminal portion of Pnut. sip2- sip4 encode proteins without obviously informative motifs. sip2 (CG9188) encodes a protein that interacted with full-length Sep1 and Sep2 but not with Pnut. sip3 (CG1937) encodes a protein that appeared to interact specifically with the C-terminal portion of Sep2. sip4 was identified independently as dip2 (Dorsal interacting protein 2) (Bhaskar, 2000); it encodes a protein that interacts with all of the Sep1 and Sep2 fusions and (weakly) with the N-terminal portion of Pnut (Shih, 2002).
Sequencing of a full-length clone of Dmuba2 showed that the predicted DmUba2 contains 700 amino acids and has 29% sequence identity to yeast Uba2p and 48% identity to human hUba2, as observed also by others (Bhaskar, 2000; Long, 2000; Donaghue, 2000). Like its homologues and other E1-type enzymes, DmUba2 contains an ATP-binding motif (amino acids 26-31) and the consensus Cys (C175) corresponding to those essential for thiolester bond formation in other E1-type enzymes. The original two-hybrid clone of Dmubc9 appeared to be full length by comparison to yeast UBC9, and the sequence for Dmubc9 agreed with that reported by Joanisse (1998). Antibodies were raised to DmUba2. The affinity-purified antibodies recognized mainly one polypeptide of apparent molecular weight ~97 kDa, which is presumably DmUba2 (predicted molecular weight, 77.5 kDa). Support for this conclusion was obtained by expressing Dmuba2 under GAL-promoter control in yeast. When cells were grown under inducing conditions, the antibodies recognized primarily a polypeptide of apparent molecular weight ~97 kDa that was absent when cells were grown under repressing conditions. Similarly anomalous low mobility on SDS-PAGE has been noted for both Uba2p and hUba2 (Shih, 2002).
In S. cerevisiae, the E1 enzyme for ubiquitin activation is the 1024 amino-acid Uba1p. In contrast, Smt3p is activated by a heterodimer of the 636 amino-acid Uba2p, which is related to the C-terminal part of Uba1p, and the 347 amino-acid Aos1p, which is related to the N-terminal part of Uba1p. Similarly, the 700 amino-acid DmUba2 is related in sequence (~40% identity over the ~225 amino acids of the three similarity boxes defined for other Uba1- type and Uba2-type enzymes to the C-terminal part of the putative Drosophila ubiquitin-activating enzyme DmUba1. Therefore, a Drosophila homologue of yeast AOS1 was identified among the ESTs from the BDGP (Shih, 2002).
Sequencing (Accession No. AF193554) showed that Dmaos1 encodes a polypeptide of 337 amino acids that has 28% sequence identity to yeast Aos1p and 40% identity to the human Aos1p homologue Sua1, as also observed by others (Bhaskar, 2000; Long, 2000). As expected, DmAos1 is related in sequence to the N-terminal part of DmUba1 (~37% identity over the ~202 amino acids of the similarity boxes as defined previously, suggesting that a heterodimer of DmUba2 and DmAos1 is the Drosophila Smt3/SUMO-activating enzyme. In support of this hypothesis, an interaction was detected between full-length DmUba2 and full-length DmAos1 in the two-hybrid system. An attempt to use the two-hybrid system to delimit the region of DmUba2 responsible for its interaction with DmAos1 was unsuccessful (Shih, 2002).
A homolog of SMT3/SUMO-1 was identified among the BDGP EST clones. The predicted DmSmt3 contains 90 amino acids with 48% identity to yeast Smt3p and 54% identity to human SUMO-1, as also observed by others (Bhaskar, 2000; Huang, 1998; Lehembre, 2000). Polyclonal antibodies were generated and immunoblots were performed on fly extracts, expecting to detect both free DmSmt3 and DmSmt3- modified proteins. Because yeast and mammalian cells contain Smt3p/SUMO-1-specific isopeptidases, which remove Smt3/SUMO from modified proteins, extracts were prepared both with and without N-ethylmaleimide (NEM), an isopeptidase inhibitor. In both extracts, the purified antibodies recognized both a polypeptide of ~16 kDa (presumably free DmSmt3) and many polypeptides of higher molecular weight (presumably DmSmt3-conjugated proteins). As expected, the higher molecular-weight species were both less abundant and of lower average molecular weight when extracts were prepared without NEM (Shih, 2002).
To test the hypothesis that DmUba2/DmAos1 and DmUbc9 are activating and conjugating enzymes for DmSmt3, but not for ubiquitin (DmUb), in vitro protein-binding assays were used to investigate the interactions among these proteins. Because ubiquitin and ubiquitin-like proteins undergo proteolytic cleavage of their C-termini to leave the sequence Gly-Gly, which is essential for both activation and conjugation, DmSmt3 and DmUb were cloned such that they terminated with Gly88 (DmSmt3) or Gly76 (DmUb) and were tagged with His6 at their N-terminal ends. Then incubated purified His6DmSmt3 and purified His6DmUb were incubated with fly extracts. The His6-tagged proteins were isolated using Ni-NTA beads, and the associated proteins were analyzed. As expected, both DmUba2 and DmUbc9 associated only with His6DmSmt3 and not with His6DmUb. The anti-DmUba2 antibodies detected not only the free form of DmUba2 (~97 kDa) but also species whose lower mobilities suggested that they might represent DmUba2 conjugated to one, two, or three molecules of DmSmt3 (and/or some other ubiquitin-like molecule). To ask if the interactions of DmUba2 and DmUbc9 with DmSmt3 involved thiolester bonds, the experiments were repeated but ß-mercaptoethanol (which reduces thiolester bonds) was omitted during sample preparation. As expected, the most abundant species now observed with the anti-DmUba2 antibodies had an apparent molecular weight of ~116 kDa, consistent with its being DmUba2 with a single His6DmSmt3 linked by a thiolester bond. Similarly, the anti-DmUbc9 antibodies now revealed an additional species with an apparent molecular weight of ~30 kDa, presumably representing DmUbc9 with a single His6DmSmt3 linked by a thiolester bond (Shih, 2002).
Immunoprecipitation was used to ask whether DmUba2 and DmUbc9 interact in vivo. When immunoprecipitates were prepared from embryo extracts using antibodies to either protein, the other protein was detected by immunoblotting. Taken together, the results of in vitro binding assays and coimmunoprecipitation suggest that DmUba2/DmAos1 and DmUbc9 are indeed the activating and conjugating enzymes, respectively, for DmSmt3. They may form a complex containing both the E1-type and E2-type enzymes (Shih, 2002).
To begin investigating the roles of the DmSmt3-conjugation pathway in Drosophila, immunofluorescence and confocal microscopy was used to characterize the intracellular localization of DmUba2. Yeast Uba2p is concentrated in the nucleus, but the localization of the homologous enzyme has not been examined in multicellular organisms. Interestingly, DmUba2 is not exclusively nuclear during early embryogenesis. Before migration of the nuclei to the embryo cortex after nuclear division 9, DmUba2 was found largely in the cortex, and its distribution there appeared homogenous. During the interphase preceding nuclear division 10, DmUba2 gradually became organized into a cap corresponding approximately to the cortical actin cap that forms over each nucleus. DmUba2 was also found in the deeper cytoplasm and gradually moved into the nuclei. During mitosis, DmUba2 was dispersed in the cortex and in the cytoplasm near the cortex. During the three subsequent nuclear cycles, the cap-like localization, progressive nuclear accumulation, and dispersion during mitosis of DmUba2 remained evident. However, during the successive cycles, the cap-like cortical localization became more organized and the degree of nuclear enrichment became more pronounced. By cycle 13, although some DmUba2 still localized to the cortex during interphase, it was predominantly nuclear (Shih, 2002).
Because DmUba2 was identified by its two-hybrid interaction with Sep1 and Sep2, whether DmUba2 colocalized with the septins either at the cellularization front or in cleavage furrows in mitotic domains after gastrulation was examined carefully. During cellularization, most DmUba2 localized to nuclei, accumulating preferentially at their apical ends. No DmUba2 was detected at the cellularization front. However, some DmUba2 remained at the cortex, where diffuse septin staining was also observed. During mitosis in older embryos, DmUba2 spread throughout the cell but did not become detectably concentrated in cleavage furrows; it then moved back into the nuclei after mitosis, with no detectable concentration at the midbody. Thus, no substantial colocalization of DmUba2 and septins was detected in early embryogenesis. However, it remains possible that DmUba2 could interact with septins at the cortex in syncytial-blastoderm embryos, during cellularization, or in mitotic cells. DmUba2 was concentrated in nuclei of nondividing cells and dispersed throughout the cell during mitosis throughout embryonic stages 6 to 15. This was particularly striking in the CNS, where the septins, in contrast, are enriched in axons (Shih, 2002).
DmUba2 localization was also examined during oogenesis. DmUba2 localized to the nuclei of both germ cells and somatic follicle cells in the germarium. After encapsulation of the germ-line cells by the follicle cells, DmUba2 remained localized to follicle cell nuclei. DmUba2 localization to nurse cells decreased as egg chamber development progressed, but it remained enriched in the oocyte nucleus. In contrast, Pnut localizes primarily to the basal surface of the follicle cells and is excluded from nuclei (Shih, 2002).
The two-hybrid interactions between the septins and DmUba2 and DmUbc9 might reflect a physiologically significant but transient interaction, such as might occur if Drosophila septins, like yeast septins, are Smt3 modified. To explore this possibility, immunofluorescence was used to examine DmSmt3 localization in cultured cells and in cellularizing and older embryos. DmSmt3 did not colocalize detectably with the septins at the cellularization front. Instead, DmSmt3 localized to nuclei, with a particular enrichment at their apical ends, as did DmUba2. In cultured cells and in cells of postgastrulation embryos, DmSmt3 was concentrated in nuclei throughout interphase. During mitosis, DmSmt3 initially appeared to spread throughout the cell. However, during metaphase, DmSmt3 appeared to concentrate in the region of the chromosomes; this was confirmed by localizing DmSmt3 relative to the mitotic spindle. Strikingly, DmSmt3 was also found concentrated in a spot at cleavage furrows and midbodies both in cultured cells and in dividing embryonic cells; this spot overlapped, but did not appear to coincide fully, with the concentration of the septins in these furrows. DmSmt3 was not enriched at the cleavage furrows early in furrow formation, but only during later stages, and it remained concentrated in the midbody after most DmSmt3, and essentially all of the DmUba2, had reaccumulated in nuclei (Shih, 2002).
DmSmt3 localization was also examined during other stages of embryogenesis. During the syncytial cell cycles, DmSmt3 was concentrated in the nuclei during interphase and appeared to localize to the chromosomes during mitosis. Like DmUba2, DmSmt3 localized primarily to the nuclei of non-mitotic cells throughout the rest of embryogenesis, including in the CNS (where the septins, in contrast, localized to axons). The concentration of DmSmt3 in late cleavage furrows and midbodies suggested that one or more of the Drosophila septins might be modified by DmSmt3. Various experimental approaches were tried to look for DmSmt3-modified septins. None of these approaches detected DmSmt3 modification of Pnut, Sep1, or Sep2. These negative results may mean that the septins are not Smt3 modified. However, immunofluorescence studies detected colocalization of Smt3 with the septins only for a brief period at the end of mitosis, and even at this stage the overlap was not complete. Thus even if septins are Smt3 modified during this period, they would probably comprise only a very small fraction of the total septins in the embryo and thus might have escaped detection (Shih, 2002).
The Drosophila septins appear to be essential for cytokinesis in at least some cell types, and it is likely that they have a variety of non-cytokinesis roles as well. Because studies in yeast suggest that a primary function of the septins is to serve as a matrix or template for the organization of other proteins at the cell surface, the identification of septin-interacting proteins should be critical to the elucidation of septin function in Drosophila. This study began with an attempt to identify such proteins using the yeast two-hybrid system. Of 27 positive clones identified with three septin baits, 17 contained fragments of the septin genes themselves. Because other evidence suggests strongly that the septins interact with each other in vivo, this result suggests that the baits used were good and that the screen was otherwise of high fidelity. Thus, it seems likely that at least some of the other positive clones represent genes whose products really interact with septins in vivo (Shih, 2002).
In conclusion everal lines of evidence suggest that the two-hybrid interactions observed between DmUba2, DmUbc9, and the septins reflect physiologically significant interactions. (1) Among the 10 positive clones that did not encode septins, four contained either Dmuba2 or Dmubc9, and Dmuba2 fragments were isolated with two different septin baits. (2) Because DmUba2 and DmUbc9 also interact with each other, the identification of both genes independently in screens using septin baits suggests that the interactions are relevant. (3) While these studies were in progress, it became clear that yeast septins are extensively modified by Smt3p, although the functional significance of that remains uncertain (Johnson, 1999; Takahashi, 1999; Takahashi, 2001; Johnson, 2001). (4) Several other groups also isolated Uba2 and/or Ubc9 in two-hybrid screens using other protein baits. Several of these interactors, including Drosophila calcium/calmodulindependent kinase II (CAM-kinase II; Long, 2000) and Dorsal (Bhaskar, 2000), were subsequently shown to be Smt3 modified (Shih, 2002).
Other genetic data may also reflect an interaction between the septins and the DmSmt3 system. The pnut septin mutation was originally identified as an enhancer of a sina mutation that affects R7 photoreceptor development. Although the significance of this genetic interaction remains unclear, it may reflect the recently discovered crosstalk between Smt3/SUMO modification and ubiquitination. Mammalian SUMO-1 is conjugated to the protein Mdm2, a RING-finger E3 ubiquitin ligase involved in p53 degradation. Modification by SUMO-1 appears to regulate Mdm2 activity and hence the level of p53, probably by regulating the ubiquitination and degradation of Mdm2 itself. Other RING-finger proteins, including Drosophila Sina, interact with Ubc9 family proteins and/or are modified by Smt3/SUMO (Duprez, 1999; Hu, 1997). Thus, it seems possible that DmSmt3 modification regulates the activities of Sina, such as its role in downregulating the transcriptional repressor Tramtrack (one of whose isoforms is itself DmSmt3 modified) (Lehembre, 2000; Li, 1997; Neufeld, 1998; Tang, 1997), and that Pnut plays a role in mediating the requisite interactions. Finally, in several types of dividing Drosophila cells, it was found that DmSmt3 colocalizes with septins in the cleavage furrows and/or midbodies during cytokinesis. Interestingly, DmSmt3 is not enriched in the furrow during the early stages of furrow formation but only later, at a time when most DmUba2 has moved back into the nuclei. Although DmSmt3 modification of Sep1, Sep2, or Pnut could not be detected (despite considerable effort), it remains possible that one or more of these proteins is modified at low levels or that DmSmt3 is conjugated to Sep4 or Sep5. However, it is also possible that the DmSmt3 in the midzone is conjugated to other proteins, such as the 'chromosomal passenger proteins,' which are associated with the chromosomes and then relocate to the spindle midzone in mitosis. The involvement of such proteins in chromosome segregation as well as in cytokinesis might help to explain the observations suggesting that the Smt3/SUMO system is involved in chromosome segregation (Apionishev, 2001; Meluh, 1995; Tanaka, 1999; Shih, 2002 and references therein).
It also remains unclear whether the Drosophila septins ever serve as a matrix/template for the localization of the DmSmt3 conjugation system. Although immunofluorescence studies show that DmUba2 and the septins are sometimes in the same compartment, so that interaction would be possible, no persuasive colocalization of the proteins was detected. Thus, elucidation of the possible interactions between the septins and the Smt3 system in Drosophila, and of their functional significance, will need to await further studies using other approaches (Shih, 2002).
Biochemical and two-hybrid studies indicate that there are multiple DmSmt3-modified proteins, that DmSmt3-specific isopeptidases probably exist, that DmUba2 and DmAos1 interact with each other, and that both DmUba2 and DmUbc9 become conjugated to DmSmt3, but not to DmUbiquitin, by thiolester bonds. While these studies were in progress, related findings were also made by other investigators who were led by other routes to the Smt3/SUMO system in Drosophila. In particular, several other proteins were shown to interact with DmUba2 and DmUbc9 using the two-hybrid system, and multiple DmSmt3-modified proteins were observed (Bhaskar, 2000; Donaghue, 2001; Joanisse, 1998; Lehembre, 2000; Long, 2000; Ohsako, 1999), supporting the hypothesis that DmSmt3 is indeed conjugated to a variety of proteins in vivo. In addition, the DmUba2/DmAos1 interaction and the conjugation of DmSmt3 to DmUba2 and DmUbc9 by thiolester bonds were also observed using other methods (Lehembre, 2000; Long, 2000). Finally, it was shown that Dmubc9 can functionally complement a yeast ubc9 mutation (Joanisse, 1998; Ohsako, 1999). Taken together, these results make clear that the Smt3/SUMO conjugation system is closely conserved between Drosophila, yeast, and mammals. The data also show that DmUba2 and DmUbc9 form a complex in vivo, suggesting that the conjugation machinery may act in a concerted fashion (Shih, 2002).
Studies in other laboratories also provided clues to possible functions of modification by DmSmt3. In particular, the semushi (Epps, 1998) and lesswright (Apionishev, 2001) mutations are both in Dmubc9, suggesting (from their mutant phenotypes) that DmUbc9 has roles in the nuclear import of the transcription factor Bicoid and in meiotic chromosome segregation. In addition, two other transcriptional regulators, Dorsal and Tramtrack, as well as CAM-kinase II, have also been shown to be modified by DmSmt3 (Bhaskar, 2000; Lehembre, 2000; Long, 2000). In the case of Dorsal, as with Bicoid, DmSmt3 conjugation appears to promote nuclear localization, whereas Tramtrack modification may help to regulate its activity and/or its degradation by the proteasome, as discussed above. The modification of CAM-kinase II may regulate its activity (Shih, 2002).
Although most suggested functions of the Smt3/SUMO system in Drosophila, as well as in yeast and mammalian cells, center on nuclear proteins, immunofluorescence and two-hybrid data support the hypothesis that there are also cytoplasmic targets. (1) It remains likely that in Drosophila, as in yeast, there is an interaction between the Smt3/SUMO system and the septins, which appear to be exclusively cytoplasmic proteins. (2) Some DmSmt3- modified proteins appear to remain both at the embryo cortex during cellularization and in the midbodies that remain after the nuclear envelopes have reformed at the end of cytokinesis. (3) Although DmUba2 and DmUbc9 (Joanisse, 1998; Lehembre, 2000) are found primarily in nuclei, considerable DmUba2 is also found in the cytoplasm and at the cortex during the syncytial-blastoderm stage, suggesting that DmSmt3 modification of cortical and/or cytoplasmic proteins could occur. (4) The cell-cycle-regulated translocation of DmUba2 between cytoplasm and nucleus both in syncytial blastoderm and in post-cellularization embryos may suggest that the partitioning of the DmSmt3-conjugation system between the cytoplasm and the nucleus is important and thus well regulated during embryonic development (Shih, 2002).
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