A Ubiquitin-Conjugating Enzyme Is Essential for Developmental...

Molecular Biology of the CellVol. 8, 1989–2002, October 1997

A Ubiquitin-Conjugating Enzyme Is Essential forDevelopmental Transitions in DictyosteliumAlexandra Clark,* Anson Nomura,* Sudhasri Mohanty,and Richard A. Firtel†

Department of Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla,California 92093-0634

Submitted February 6, 1997; Accepted July 16, 1997Monitoring Editor: James A. Spudich

We have identified a developmentally essential gene, UbcB, by insertional mutagenesis. Theencoded protein (UBC1) shows very high amino acid sequence identity to ubiquitin-conju-gating enzymes from other organisms, suggesting that UBC1 is involved in protein ubiq-uitination and possibly degradation during Dictyostelium development. Consistent with thehomology of the UBC1 protein to UBCs, the developmental pattern of protein ubiquitinationis altered in ubcB-null cells. ubcB-null cells are blocked in the ability to properly execute thedevelopmental transition that occurs between the induction of postaggregative gene expres-sion during mound formation and the induction of cell-type differentiation and subsequentmorphogenesis. ubcB-null cells plated on agar form mounds with normal kinetics; however,they remain at this stage for ;10 h before forming multiple tips and fingers that then arrest.Under other conditions, some of the fingers form migrating slugs, but no culmination isobserved. In ubcB-null cells, postaggregative gene transcripts accumulate to very high levelsand do not decrease significantly with time as they do in wild-type cells. Expression ofcell-type-specific genes is very delayed, with the level of prespore-specific gene expressionbeing significantly reduced compared with that in wild-type cells. lacZ reporter studies usingdevelopmentally regulated and cell-type-specific promoters suggest that ubcB-null cells showan unusually elevated level of staining of lacZ reporters expressed in anterior-like cells, aregulatory cell population found scattered throughout the aggregate, and reduced staining ofa prespore reporter. ubcB-null cells in a chimeric organism containing predominantly wild-type cells are able to undergo terminal differentiation but show altered spatial localization. Incontrast, in chimeras containing only a small fraction of wild-type cells, the mature fruitingbody is very small and composed almost exclusively of wild-type cells, with the ubcB-nullcells being present as a mass of cells located in extreme posterior of the developing organism.The amino acid sequence analysis of the UbcB open reading frame (ORF) and the analysis ofthe developmental phenotypes suggest that tip formation and subsequent developmentrequires specific protein ubiquitination, and possibly degradation.

INTRODUCTION

A significant amount of information has been obtainedabout the signal transduction pathways that controlmany aspects of Dictyostelium differentiation (Dev-reotes, 1994; Firtel, 1995). Cyclic AMP, functioning as

an extracellular ligand to a seven-span/serpentineclass of receptors, mediates aggregation, the develop-mental switch to multicellular development, and cell-type differentiation through distinct signal transduc-tion pathways. Subsequent morphogenesis and theinitiation of culmination are also believed to be regu-lated by cAMP functioning both as an extracellularsignaling molecule and intracellularly through the ac-tivation of PKA (Williams and Morrison, 1994; Firtel,1995). While both molecular and biochemical ap-

* These two authors contributed equally to this work.† Corresponding author: Center for Molecular Genetics, Room

225, University of California, San Diego, 9500 Gilman Drive, LaJolla, CA 92093-0634.

© 1997 by The American Society for Cell Biology 1989

proaches have resulted in significant insights intothese and other pathways controlling Dictyosteliumdifferentiation, it is clear that many other regulatorynetworks participate in controlling differentiation ofthis organism.

Genetic analysis in other systems has identifiedubiquitination and deubiquitination as importantmechanisms controlling a diversity of cellular regula-tory processes, including ubiquitin-mediated proteinturnover, protein targeting, and cell fate decisions.Ubiquitin conjugating enzymes UBC/E2 are thoughtto be involved in substrate recognition: the multiplic-ity of UBC identified in a number of organisms sug-gests that each UBC may be important in controllingthe ubiquitination of a specific subset of proteins (Her-shko and Ciechanover, 1992; Varshavsky, 1992; Ciec-hanover, 1994). For example, a mammalian UBC/E2protein is involved in p53 degradation; UBC6 andUBC7 are involved in degrading MATa2 in yeast;UBC2 has been implicated in degradation of the Gaprotein subunit GPA1 in yeast; UBC9 is implicated incyclin degradation essential for the progression of thecell cycle in yeast; the Huntingtin protein interactswith hE2; PAS2 is required for peroxisome biogenesis;and the Drosophila bendless gene is involved in synaptictransmission (Dohmen et al., 1991; Glotzer et al., 1991;Wiebel and Kunau, 1992; Chen et al., 1993; Muralidharand Thomas, 1993; Chowdary et al., 1994; Ciechh-anover et al., 1994; Madura and Varshavsky, 1994; Ohet al., 1994; Palombella et al., 1994; Seufert et al., 1995;Kalchman et al., 1996). In addition, ubiquitination hasbeen shown to be a component of the pathway bywhich the yeast MDR transporter Pdr5, the yeast a-factor receptor, and the mammalian growth hormonereceptor are targeted for endocytosis (Egner andKuchler, 1996; Hicke and Riezman, 1996; Strous et al.,1996), while ubiquitination/deubiquitination controlssome cell fate decisions during development of theDrosophila eye (Jermyn and Williams, 1995) and larvaldevelopment in Caenorhabditis elegans (Zhen et al.,1996). In Dictyostelium, the structure and regulation ofexpression of genes encoding ubiquitin have been ex-amined (Muller-Taubenberger et al., 1988a and 1988b;Ohmachi et al., 1989), and the in vitro ubiquitinationand degradation of Dictyostelium calmodulin havebeen reported in a reticulocyte extract (Gregori et al.,1985). However, little is know about the role of proteinubiquitination during Dictyostelium development.

In this article, we report the characterization of thegene UbcB that, when disrupted, results in develop-mental arrest during the multicellular stages of devel-opment. UbcB encodes a putative ubiquitin-conjugat-ing enzyme designated UBC1. Disruption of UbcBaffects tip formation, cell-type differentiation, the abil-ity of ubcB-null cells to participate in normal develop-ment in chimeras containing wild-type cells, and thepattern of protein ubiquitination during development.

Our results suggest that protein ubiquitination at themound and finger stage mediated by UBC1 is requiredfor subsequent development and may be an essentialcomponent of the developmental transition betweenthe induction of postaggregative gene expression andsubsequent cell-type differentiation and morphogene-sis.

MATERIALS AND METHODS

REMI mutagenesis using uracil selection, DH1 uracil auxotroph,and the pRHI30 vector, was done as previously described (Kuspaand Loomis, 1992; Dynes et al., 1994; Insall et al., 1994). Cell culture,transformation, and selection of clones expressing lacZ reporterswere performed as previously described (Dynes et al., 1994; Gaskinset al., 1996). All other techniques, including the isolation and cloningof the insertion vector from the REMI mutant, construction of vec-tors, sequencing, analysis, and RNA, Southern, and Western blottechniques are standard and were performed as described previ-ously (Dynes et al., 1994; Gaskins et al., 1994, 1996). Standard South-ern hybridization conditions for Dictyostelium genomic DNA, whichis AT-rich, are 33 SSC, 60 mM sodium phosphate (pH 6.8), 10 mMEDTA (pH 7.2), 0.2% SDS, 43 Denhardt’s solution, and 50%deioinized formamide at 37°C as described previously (Nellen et al.,1987). For lower stringency hybridizations (see text), the salt con-centrations were kept constant during the hybridization; however,the salt concentrations in the wash solutions were doubled and thelast wash of 5 mM EDTA, pH 7.2, and 1% SDS was replaced with 13SSC, 1% SDS.

Western blot analysis was performed as described previously(Gaskins et al., 1994).

RESULTS

Isolation of UbcB and Identification of TranscriptsWe identified UbcB (see below) as a developmentallyessential gene in a REMI mutagenesis (Kuspa andLoomis, 1992) screen for genes required for multicel-lular development. REMI mutagenesis was performedusing an insertion vector (pRHI30) carrying the Dic-tyostelium Pyr5–6 gene (uracil prototroph) trans-formed into the uracil auxotroph DH1, selecting foruracil prototrophs (Kuspa and Loomis, 1992; Insall etal., 1994). The insertion vector was mapped to a singlesite in the Dictyostelium genome by Southern blot anal-ysis (Figure 1A; our unpublished observations). Therewas an apparent shift in the mobility of the endoge-nous UbcB band in the mutant strain, consistent witha UbcB gene disruption. The vector and ;1.7 kb offlanking DNA were excised by digestion with therestriction enzyme EcoRI, circularized by ligation, andcloned into Escherichia coli (see MATERIALS ANDMETHODS). Restriction maps of the genomic andcDNA clones (see below) are shown in Figure 2, A andB. To confirm that the developmental phenotype re-sulted from insertion at this locus, we retransformedthe isolated DNA into Dictyostelium after linearizing itby digestion with EcoRI. Approximately 60% of theclones had the same developmental phenotype as theoriginal REMI mutation, while the remainder were

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Molecular Biology of the Cell1990

wild type. Southern blot analysis showed a 1:1 corre-lation with the phenotype of the original REMI strain(see below) and insertion of the DNA at the same site(our unpublished observations).

DNA from both sides of the insertion site of thegenomic clone hybridized to two transcripts of ;0.65and 0.75 kb (Figure 2C; our unpublished observa-tions). The larger transcript was expressed duringgrowth and development, with levels decreasing inthe multicellular stages. The smaller transcript wasexpressed at very low levels during vegetativegrowth, and the levels increased during the time ofaggregation. This transcript was present throughoutthe remainder of development. When the ubcB mutantstrain is allowed to develop for 8 h (early moundformation) and RNA is isolated and hybridized toeither probe, no hybridization is observed, implyingthat both transcripts are encoded by the same gene(our unpublished observations). From the lack of de-tectable transcripts in the mutant strain, we concludethis is an ubcB-null strain. Expression of the UbcBcDNA downstream from the Act15 promoter comple-mented the null phenotype.

Hybridization probes were then used to screen alZap library (Schnitzler et al., 1994). Several sets ofcDNA clones ranging from 0.6 to 0.8 kb were identi-fied. The sequence of a larger and smaller clone

showed that both encoded the same open readingframe (ORF) frame but had different lengths of 59 and39 untranslated sequences. This suggests that differ-ences in the two transcripts probably result from dif-ferent start and/or termination sites and are not dueto splicing variants within the ORF (see below). More-over, the different temporal patterns of expression ofthe two RNAs suggest that UbcB transcription is reg-ulated in a complex manner.

UbcB encodes a putative ubiquitin-conjugatingenzymeThe two cDNAs of different lengths were completelysequenced in both directions and a single full-lengthORF was identified (accession number U67838). Com-parison of the sequence to the GenBank databasesusing the BLAST program showed that the UbcB ORFhas very high amino acid sequence identity to E2/ubiquitin-conjugating enzymes from yeast, variousmetazoans, and plants (Figure 3). The extremely highamino acid sequence conservation throughout the en-tire protein strongly suggests that UbcB encodes aubiquitin-conjugating enzyme. Because multiple ubiq-uitin-conjugating enzymes have been identified in avariety of other species, we expect that Dictyosteliumalso contains a complex multigene family. We have

Figure 1. Southern blot of wild-type and ubcB-null cells. Genomic DNA isolated from wild-type cells and ubcB-null cells was digested withthe restriction enzymes shown and analyzed by Southern blot hybridization as described previously at different hybridization stringencies(Nellen et al., 1987). (A, 50% formamide) The solid circle marks the proper UbcB gene band for this digest. (B, 40% formamide) The two panelsare different exposures of the same filter. (C, 33% formamide) The same batch of restriction enzyme-digested DNA was used for eachhybridization. The two 40% and 33% formamide panels are different exposures of the same filter. The positions of size markers (in kb) areshown. See MATERIALS AND METHODS for details. The open arrowhead marks the position of a common band (wild-type HincII band).The asterisks mark the positions of some bands not seen in the high stringency (50% formamide) hybridization blot but which appear in thelower stringency hybridizations.

Requirement of a UBC for Dictyostelium Development

Vol. 8, October 1997 1991

thus designated the protein encoded by UbcB asUBC1.

To investigate the possibility of additional genesencoding UBC in Dictyostelium, we performedSouthern blot analysis of DNA from wild-type andubcB-null cells under different stringencies. Asshown in Figure 1A, hybridization under standardconditions (see MATERIALS AND METHODS)identifies a single UbcB gene in wild-type cells.When the stringency is reduced by lowering theformamide concentration from 50% to 40% whilekeeping the salt concentration and hybridizationand wash temperatures constant (Figure 1B), addi-tional hybridizing bands are observed, suggestingthe presence of genes that are related at the level ofDNA sequence. When the stringency was reducedfurther (33% formamide, constant salt and temper-ature), some additional strong bands and a signifi-

cant amount of apparently nonspecific backgroundhybridization are observed (Figure 1C). The pres-ence of additional bands that are observed in the40% formamide hybridization and are stronger at33% formamide is consistent with the possibilitythat there are multiple UbcB-related genes in Dictyo-stelium.

Developmental Phenotype of ubcB-Null CellsTo examine the developmental morphologic pheno-type of the ubcB-null mutant, cells were grown ax-enically, washed of nutrients, and then plated formulticellular development on sodium/postassiumphosphate-containing agar plates. ubcB-null cellsaggregate and form a mound at 7– 8 h of develop-ment, ;1 h earlier than the wild-type parental ax-enic strain. At the same plating densities, the aggre-gate sizes of the mutant and wild-type strains wereapproximately the same [our unpublished observa-tions; Figure 4, compare panels C (wild type) and D(ubcB null)]. Wild-type mounds start to form tips at10 –11 h (Figure 4C) and begin to culminate by 18 h(Figure 4, A and B), forming a fruiting body at ;24h (Figure 4J). In contrast, ubcB cells arrest at themound stage for ;10 –12 h (Figure 4D), whereupondevelopment reinitiates with the formation of mul-tiple tips on the same aggregate (Figure 4, E and F).These tips elongate to form fingers with a majorityof the cells remaining in a mound and not partici-pating in finger formation. Development then ar-rests under these conditions at ;36 h with no mi-grating slugs being formed (Figure 4, H and I). At ahigher cell density, not all of the cells aggregate. Themounds that form also have multiple tips and pro-trusions that often produce aerial tangles (Figure4G). When cells are plated on Millipore filters, mul-tiple fingers arise from each mound after a delay of;10 –12 h. These fingers comprise only approxi-mately one-half of the cells in the original mound,with the remainder found as an undifferentiatedmass (see below; Figure 7B, panel B). At ;30 –36 h,some of the fingers form migrating slugs; however,none of these initiate culmination. When cells fromterminal structures plated on Na/K agar or Milli-pore filters are disaggregated after 36 and 48 h,respectively, and assayed for heat and detergent-resistant spores, no viable cells are found. Expres-sion of the UbcB cDNA from the Act15 promotercomplements the developmental phenotypes of theubcB-null cells, consistent with the developmentalabnormalities being the result of a disruption of theUbcB gene (our unpublished observations). Overex-pression of the cDNA in wild-type cells does notresult in any observable morphologic defect (ourunpublished observations).

Figure 2. Restriction maps of the UbcB genomic clone and thecDNA ORF and RNA blot hybridization. (A) The map of genomicclone shows the positions of the UbcB ORF translation initiation site(ATG) and termination site (TAA). Restriction sites for the genomicclone: E, EcoRI; D, DpnII; RV, EcoRV; C, ClaI; HP, HpaI. The trans-formation vector is RHI30 carrying the Pyr5–6 gene required foruracil prototrophy. The parental strain is the uracil prototroph DHI.The vector and strain were gifts from R. Insall and P. Devreotes.RF31 and T7 are sequencing primers. RF31 was obtained from W.Loomis. (B) The map of cDNA clones shows the position of theUbcB ORF translation initiation site (ATG) and termination site(TAA). The DpnII insertion site for RHI30 in the UbcB REMI mutantstrain is shown. (C) Developmental RNA blot of UbcB expression.Time in development is given. Veg, vegetative cells.

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Changes in the Pattern of Protein Ubiquitination inubcB-Null CellsBecause UbcB encodes a putative UBC, we have inves-tigated potential changes in the pattern of ubiquiti-nated proteins during development on Western blotsusing an anti-ubiquitin antibody. While most of thepattern of ubiquitinated proteins is similar betweenwild-type and ubcB-null cells (Figure 5), three proteinsthat react strongly with the antibody show significantdifferences between the two strains. In wild-type cells,a protein of ca. 73 kDa (Figure 5, band a) from itsmobility reacts weakly with the antibody through 12 hof development, the time at which the ubcB phenotypeis first observed. Afterward, the intensity of a band ofthe same mobility (and thus possibly the same pro-tein) increases, suggesting that this protein undergoeseither a reduction in the rate of degradation and/or anincrease in the level of ubiquitination and/or expres-sion. Our observations cannot exclude the possibilitythat band a represents two proteins of the same elec-trophoretic mobility but with different developmentalkinetics. In contrast with observations in wild-typecells, in the mutant strain, this band does not decreasein intensity at 12 h but shows strong staining through-out development. Another protein (band b, ca. 59 kDa)shows a reciprocal staining pattern in wild-type cells:it is detected through 12 h of development cells, afterwhich the level of the protein or the level of ubiquiti-nation of the protein rapidly declines. In ubcB-nullcells, the band continues to stain strongly for a longertime in development. A third protein (band c, ca. 51

kDa) increases in staining intensity at 4 h and remainshigh throughout the remainder of development inwild-type cells. The staining is generally weaker inubcB-null cells and the level of staining decreases afterthe 12-h time point. The level of ubiquitination ofother proteins may also be affected but is not detect-able on our blots. While these data do not directlyaddress whether any of these proteins are direct sub-strates of UBC1, they indicate that ubcB-null cellsshow observable changes in the quantitative and/orqualitative pattern of protein ubiquitination as com-pared with wild-type cells (see DISCUSSION).

Control of Gene Expression in ubcB-Null OrganismsTo better understand regulatory changes that might beoccurring in the ubcB-null cells, we examined the de-velopmental regulation of genes preferentially ex-pressed during multicellular development. The ex-pression of LagC and CP2/cprB was used as amolecular marker for postaggregate gene regulationduring mound formation (Pears et al., 1985; Datta et al.,1986; Dynes et al., 1994). In wild-type cells, both genesare induced by a high, constant level of extracellularcAMP that is thought to occur as the mound forms(Abe and Yanagisawa, 1983; Firtel, 1995). CP2/cprBencodes a cysteine protease and has been used as amarker for postaggregate gene expression (Pears et al.,1985; Datta et al., 1986). LagC, which is similarly reg-ulated, encodes a putative transmembrane proteinthat is essential for development past the mound stage

Figure 3. Amino acid sequence of the UbcB ORF and comparison to UBCs from a variety of organisms. Names and accession numbers aregiven below. UBC1, this article; ScUBC5, Saccharomyces cerevisiae UBC5, acccession no. P15732; HsUBC2, human UBC2, accession no. P35129;CeUBC2, C. elegans UBC2, accession no. P35129; ScUBC4, S. cerevisiae UBC4, accession no. P15731; DmUBC1, Drosophila melanogaster UBC1,accession no. P25867; ScUBC5, S. cerevisiae UBC5, accession no. P15732; AtUBC1, Arabidopsis thaliana UBC1, ac. # P25865; RnUBC, rat,accession no. P23567; HsHHR6A, human UBC, accession no. P06104; ScUBC21, S. cerevisiae UBC21, accession no. P06104; ScUBC1, S. cerevisiaeUBC1, accession no. P21734; ScUBC8, S. cerevisiae UBC8, accession no. P28263; Dmben, D. melanogaster Bendless (UBC), accession no. P35128.



and is required for prestalk and prespore cell differ-entiation (Dynes et al., 1994). In wild-type cells, CP2/cprB and LagC transcripts start to accumulate late inaggregation as the mound forms. Transcript levelsremain high for 4–6 h through the tipped aggregateand first finger stages (;11–13 h), after which levels ofthe transcripts decrease significantly (Figure 6). InubcB-null cells, both genes are induced during moundformation, but in contrast to observations in wild-typecells, the level of transcript accumulation is .10-foldhigher and remains high for ;10 h, until the timeubcB-null cells start to form tips, before decreasingonly moderately. This extended expression of CP2/cprB and LagC is consistent with the developmentaldelay of the mutant at the mound stage and its sub-

sequent arrest at the finger stage. The high, extendedlevel of expression suggests that the regulatory mech-anism involved in down-regulating these genes dur-ing finger formation is blocked in ubcB-null cells.

Analysis of the expression of the endogenousprestalk-specific gene ecmA demonstrated that thegene was induced ;12 h later in ubcB-null cells thanwild-type cells and showed a similar level of expres-sion in both wild-type and ubcB-null strains (Figure 6).The time of ecmA induction corresponds approxi-mately to the time of tip formation in the mutantstrain. Expression of the prespore-specific gene SP60/cotC shows a similar delay in induction, indicatingthat the onset of expression of both prestalk and pre-spore genes is equally affected. However, in contrast

Figure 4. Developmental morphology of ubcB-null cellsplated on Na/K P04 agar plates. (A–B) Wild-type cells atthe second finger/early culmination stage (18 h). (C) Wild-type tight aggregates forming tips (12 h), which are indi-cated by arrowheads. Note that each aggregate has onlyone tip. (D) ubcB-null cells at 18 h. Mounds first form at ;8h. Notice that no tips are present. (E) ubcB-null cells at 19 has tips are first forming. (F) Enlargement of the lower leftaggregate in E. The multiple tips are indicated by arrow-heads. (G) ubcB-null cells plated at a higher density at 36 h.(H–I) ubcB-null cells at 36 h. (J) Mature wild-type fruitingbody (26 h).

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to ecmA expression, SP60/cotC mRNA accumulation inubcB-null cells is significantly lower than in wild-typecells.

Spatial Patterning of Prestalk and Prespore Cells inubcB-Null Cells and ubcB-Null/Wild-Type CellChimerasThe effect of the ubcB-null mutation on spatial pattern-ing of prestalk and prespore cells in the multicellularorganisms was examined using cell-type-specific lacZreporters and histochemical staining. Several cell-type-specific and cell-type-enriched reporters were ex-amined. The cloned promoter of the prestalk-specificgene ecmA has been dissected into two domains bydeletion analysis using whole-mount histochemicalstaining of lacZ reporters to define spatial patterninginto regulatory elements that are preferentially ex-pressed in prestalk A and AB cells and in prestalk Oand anterior-like cells (ALC), respectively (Early et al.,1993, 1995). The vector containing the entire promoterdriving lacZ (ecmAO/lacZ) exhibits maximal expres-sion in the anterior prestalk A and AB cells and isexpressed at a lower level in prestalk O cells in theslug. It is also expressed in ALC (Early et al., 1993), aregulatory cell population comprising ;5–10% of thecells that are scattered throughout the slug. Theprestalk gene ecmB is expressed primarily in a centralcore of prestalk AB cells localized in the very anteriorof the slug but is also expressed in the ALC population(Jermyn et al., 1989; Jermyn and Williams, 1991). rasDis a prestalk-enriched gene that is expressed predom-inantly in prestalk A, prestalk O, and ALC in the slug(Esch, 1991), while SP60/cotC is a prespore-specificgene expressed in the posterior 80% of the slug(Haberstroh and Firtel, 1990). Figure 7A shows thestaining pattern of ecmAO/lacZ, SP60/lacZ, ecmB/lacZ,and rasD/lacZ in wild-type slugs.

When ecmAO/lacZ- and ecmB/lacZ-expressing ubcB-null cells were examined, little or no staining wasobserved until ;15 h after the time of mound forma-tion (our unpublished observations). In ubcB-null mi-grating slugs, the spatial pattern of ecmAO/lacZ ex-pression was similar to that seen in wild-type cells(Figure 7B, panels E and F). ecmB expression, however,was abnormal. Staining is significantly stronger thanin wild-type cells at similar copy numbers of the re-porter vector and is observed throughout the devel-oping first finger slug (Figure 6B, panel A; our unpub-lished observations). This may be an indication of anincrease in the level of staining in each expressing cellor an increase in the number of ALC. Similarly, whenrasD was examined, ubcB-null mounds produced afinger in which almost the entire finger was stainedintensely (Figure 7B, panel B), instead of the anterior,prestalk zone staining that is seen in wild-type cells atthis same stage (Esch and Firtel, 1991; our unpub-

lished observations). Cells remaining in the moundthat did not participate in finger formation showedonly scattered staining. In the migrating slug, the veryanterior of the slug stained the strongest (prestalk Adomain) but ubcB-null slugs also showed strong stain-ing through the posterior of the slug (Figure 7B, panelC). Wild-type slugs expressing rasD/lacZ showedstrong staining in the prestalk A/O region but only

Figure 5. Anti-ubiquitin Western blot. (A) Cells were taken at thetimes in development indicated, lysed in PAGE-SDS gel buffer,sized on a 10% gel, blotted to Immobilon-P membrane (Millipore),and probed with a commercial antiubiquitin antibody (Sigma, St.Louis, MO). The position of the size markers are shown. Band a, ca.;73 kDa; band b, ca. ;59 kDa; band c, ca. ;51 kDa. (B) Enlargementof the section of the blot containing bands a–c.

Figure 6. RNA blot hybridization of reporter genes during devel-opment. Expression of developmentally regulated genes in wild-type and ubcB-null strains: LagC and CP2/cprB, postaggregativegenes; ecmA, prestalk-specific; SP60/cotC, prespore-specific. The fil-ters assaying the expression level of the same gene were probedtogether.



light scattered staining of ALC in the prespore regionand moderate staining of the posterior-most rearguardzone (Figure 7A, panel D; Esch and Firtel, 1991), whichforms the basal disk in the mature fruiting body. Asimilar pattern was observed for all clonal isolatesstudied. As rasD/lacZ stains predominantly prestalkand ALC in wild-type slugs (Esch and Firtel, 1991), themore intense staining in the posterior may indicate amore intense staining of the ALC or an increase in thenumber of ubcB-derived ALC. It has been suggestedthat rasD is initially expressed in all cells and then theexpression becomes significantly weaker in presporecells (Jermyn and Williams, 1995). However, the ob-served rasD/lacZ staining pattern cannot simply bedue to a continued expression of rasD in all ubcB-nullcells, as there is negligible staining in the cells left inthe mound.

When the prespore-specific marker SP60/cotC wasexamined, staining was seen in the posterior 80% ofthe slug, as in wild-type cells (Figure 7B, panel D).However, the staining was weaker in the mutant com-pared with that in wild-type cells. This pattern wasalso reproducible in multiple clones from independenttransformations. When wild-type and ubcB-null slugswere dissociated into single cells and the number of

prespore cells counted (SP60/lacZ staining cells), asimilar fraction of cells stained in both strains (ourunpublished observations), indicating that the num-ber of prespore cells in the slugs is not altered signif-icantly in the ubcB-null mutant.

To obtain a better understanding of the properties ofthe ubcB cells, experiments were performed in whichlacZ-tagged ubcB-null and wild-type cells were mixedin a ratio of 1:3 and allowed to coaggregate, formingchimeric organisms. These chimeras were then histo-chemically stained for b-galactosidase activity for lo-calization of lacZ-expressing ubcB-null cells. Suchstudies allow the examination of both the spatial lo-calization of the ubcB cells and the ability of the cells toparticipate in different terminal structures of the fruit-ing body. When ecmAO/lacZ and ecmB/lacZ expressionwas examined in the chimeric slug, no staining wasdetectable (our unpublished observations). For rasD/lacZ, staining was significantly less intense comparedwith that seen in ubcB-null slugs. The stained cellswere concentrated in the posterior of the slug, and nostaining was detected in the anterior prestalk region(Figure 8B). When SP60/lacZ was examined, stainingwas seen in the prespore zone in migrating slugs(Figure 8C), but in second fingers at the onset of

Figure 7. Spatial patterning of cell types in wild-type and ubcB-null cells. (A) Staining of wild-type cells at the slug stage with: panel A,SP60/lacZ (prespore); panel B, ecmAO/lacZ (prestalk A, AB, and O and ALC); panel C, ecmB/lacZ (prestalk AB and ALC). Arrowhead pointsto the core of prestalk AB cells. Panel D, rasD/lacZ (prestalk and ALC). (B) Pattern of lacZ reporter staining of ubcB-null slugs using variousreporters: panel A, ecmB; panel B, rasD; panel C, rasD; panel D, SP60; panel E, ecmAO; F, ecmAO.

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culmination, staining was restricted to the posteriorhalf of the prespore zone (our unpublished observa-tions). The Act15/lacZ reporter driven by the Actin 15(Act15) promoter is expressed in all cell types (Mannand Firtel, 1991). At the slug stage, Act15/lacZ stainingwas found predominantly in the posterior 50% of theslug with the level of staining, and thus the proportionof ubcB-null cells, decreasing toward the anterior ofthe slug (Figure 8A). During early culmination, stain-ing was seen almost exclusively in the posterior 70%of the chimera, indicating ubcB cells were being ex-cluded from the anterior part of the prespore andprestalk zones (Figure 8, D and E).

In the mature fruiting body the ubcB cells were, asdetermined from Act15/lacZ staining, found in thebasal disk, lower portions of the stalk, and the lowercup and lower regions of the sorus, but were absentfrom the tip (Figure 8H). Much of the staining seen onthe sorus represents surface staining of a layer that isprobably an extension of the lower cup and derivedfrom the ALC population. When the sorus was disso-ciated, only ;8% of the spores stained (our unpub-lished observations). In mature fruiting bodies, ecmAO/lacZ staining is observed in the basal disk, the lowerportion of the stalk, and the lower cup, but no stainingwas observed in the upper cup and little staining was

Figure 8. Localization of ubcB-null cells expressing lacZ reporters in chimeras of wild-type and ubcB-null cells (ratio of 3:1). Wild-type KAx-3cells and ubcB-null cells expressing various reporters were mixed in a ratio of 3:1 and allowed to develop. Multicellular organisms werestained at the slug/finger stages (A–E) and culminants (F–K). Act15, panels A, D, E, and H; rasD, panels B and I; SP60, panels C and G. ecmAO,panel F (no staining is seen in the slug stage); ecmBD89, panel J; SpiA, panel K.



seen in the upper portion of the stalk tube (Figure 8F).rasD, which displays a similar staining pattern inwild-type fruiting bodies to that of ecmAO, alsoshowed a similar pattern in the ubcB chimeras as theecmAO/lacZ reporter with no staining seen in the up-per cup and tip, a strongly staining region in wild-type organisms (Figure 8I). lacZ expressed from theecmBD89 stalk-cell-specific promoter (Ceccarelli et al.,1991) had a similar pattern of expression: staining cellswere more concentrated in the basal disk and lowerstalk than throughout the stalk (Figure 8J). Some stain-ing was also seen in the lower cup. SP60/lacZ and lacZexpressed from the spore cell-specific promoter SpiA(Richardson et al., 1994) showed staining localized tothe lower portion of the sorus in contrast to expressionthroughout the entire sorus, as seen in wild-typestrains (Figure 8, G and K, respectively). The absenceof ubcB-null cells in the upper cup or the upper part ofthe stalk tube may be due to the absence of ubcB-nullcells in the anterior of the chimera.

To determine whether a smaller proportion of wild-type cells is able to direct the differentiation of ubcBnulls in chimeras, we mixed ubcB-null cells with wild-type cells carrying either the ecmAO/lacZ or SP60/lacZreporter in ratios of either 3:1 or 9:1 (ubcB:wild type).As shown in Figure 9 (A and B, panels A-C), even asmall proportion (10%) of wild-type cells is able todirect tip formation in a chimeric organism. However,most, if not all, of the wild-type cells appear to belocalized in the very anterior of the organism as ob-served in the panels in which the wild-type cells aretagged with Act15/lacZ, which marks all wild-typecells (Figure 9, A and B, panel A). This is also observedwhen the wild-type prespore cells are marked bySP60/lacZ. As is seen in the panel showing the 3:1 ratiomix, the SP60/lacZ expressing cells are found as a bandimmediately posterior to the position of the wild-typeprestalk cells identified by the ecmAO/lacZ marker(Figure 9A, panel E). No ecmAO/lacZ expressing cellsare observed in the posterior bulk of the chimera,which also does not show significant staining with theAct15/lacZ reporter except at the very posterior of theorganism. These results indicate that the wild-typecells are localized predominantly in the anterior of thechimera (Figure 9, A and B, panel A). When the ubcB-null cells were tagged with Act15/lacZ, no ubcB-nullcells were observed in the anterior of slugs that con-tained the wild-type cells in either the 3:1 or 9:1 ubcB-null:wild-type chimeric organisms (our unpublishedobservations).

In the final structure (Figure 9, A and B, panels D-F),the wild-type cells appear to form a fruiting body withlittle participation of the ubcB-null cells and ,1% ofthe mature spores derived from the ubcB-null strain.The patterns are very similar for 3:1 and 9:1 cell ratioswith the patterning and morphology being more se-vere in the chimera containing only 10% wild-type

cells. A very small anterior structure is seen, whichdoes not appear to contain the spore cells, as it is notlabeled with the SP60/lacZ reporter. These may beALC or prestalk cells that have not entered the stalktube, as they stain strongly with ecmAO/lacZ (Figure 9,A and B, panels E). In contrast, in many of the matureorganisms, a central swelling in the stalk appears tocontain SP60/lacZ-expressing cells (Figure 9, A and B,panels F). These data are consistent with a modelproposing that as the fraction of wild-type cells in thechimera decrease, the wild-type cells are unable toorchestrate the differentiation of the mutant cells, sug-gesting that the ubcB-null phenotype is predominantlycell autonomous.

DISCUSSION

UbcB Protein Is Required for DevelopmentalTransitions during Multicellular DevelopmentWe have identified a developmentally essential gene,UbcB, that is required at two stages during multicel-lular differentiation. ubcB-null cells form mounds andremain at this stage for ;10 h before the moundsdevelop multiple tips and then finally arrest at thefinger stage when plated on agar. When the cells areplated on Millipore filters, development proceedsslightly further, with a fraction of the fingers formingmigrating slugs. In both cases, not all of the cellsparticipate in finger and slug formation and under theconditions in which cells are plated on agar, most ofthe cells remain in the mound. The nature of thesecells is unclear. They do not stain efficiently withrasD/lacZ or cell-type-specific lacZ reporters, suggest-ing that these cells arrest before or very early duringcell-type differentiation. The observation that ubcB-null cells are able to form tips and fingers after anextended delay at the mound stage indicates that theyare eventually able to bypass the initial block at themound stage. The pathway required for tip formationmay proceed, albeit very slowly, without the pro-cesses mediated by the UbcB protein, which we havedesignated UBC1. Alternately, another ubiquitin-con-jugating enzyme may substitute for UBC1 function ata low efficiency at the mound stage during tip forma-tion, but cannot substitute later in development afterfinger/slug formation. Low stringency hybridizationsuggests the presence of additional genes encodingUBCs in Dictyostelium, which would be consistentwith published data showing that in organisms rang-ing from yeast to humans in which UBCs have beenidentified, multiple UBCs are found (see INTRODUC-TION).

In wild-type cells, postaggregative genes such as thoseencoding CP2 and LagC proteins are maximally ex-pressed at the mound stage, after which their expressiondecreases. The higher level and extended time of expres-

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sion of these two genes in ubcB-null cells suggest thatthere is a regulatory mechanism in wild-type cells, lead-ing to the down-regulation of both of these genes after

the mound stage and that this is absent in the ubcB-nullcells. The delay of induction of prestalk and presporecell-type-specific genes in ubcB cells suggests that the

Figure 9. Localization of ubcB-null cells expressing lacZ reporters in chimeras of ubcB-null and wild-type cells at ratios of 3:1 and 9:1. (A)ubcB-null and wild-type cells a ratios of 3:1. Panel A, slug stage, Act15/lacZ; panel B, slug stage, ecmAO/lacZ; panel C, slug stage, SP60/lacZ;panel D, final structure, Act15/lacZ; panel E, final structure, ecmAO/lacZ; panel F, final structure, SP60/lacZ. (B) ubcB-null and wild-type cellsat ratios of 9:1. Panel A, slug stage, Act15/lacZ; panel B, slug stage, ecmAO/lacZ; panel C, slug stage, SP60/lacZ; panel D, final structure,Act15/lacZ; panel E, final structure, ecmAO/lacZ; panel F, final structure, SP60/lacZ. In A and B, panels D-F, closed arrowheads point toenlarged region on stalk that does not stain with the prestalk/stalk marker (ecmAO/lacZ) but does stain with the prespore/spore marker(SP60/lacZ). The open arrowhead points to a small “sorus-like” structure. In A, panel C, the open arrowhead points to a wild-type presporedomain; the closed arrowhead points to a wild-type prestalk domain.



developmental arrest at the mound stage occurs be-tween the induction and down-regulation of the postag-gregative genes and the induction of the cell-type-spe-cific genes. UbcB function is essential for thedevelopmental transition required for the down-regula-tion of postaggregative gene expression.

ubcB-null cells show normal spatial patterning ofprespore cells in slugs, although the intensity of thestaining is reduced, consistent with the lower level ofexpression of the endogenous SP60/cotC gene, indicat-ing prespore gene expression is affected. The ecmAO/lacZ staining pattern also appears similar to that ofwild-type cells; however, for ecmB/lacZ- and rasD/lacZ-expressing cells, the level of their expression is veryhigh and/or an unusually high number of cells ex-press these two reporters in comparison to what isobserved in wild-type cells. The high level of stainingwith both ecmB/lacZ and rasD/lacZ suggests that theubcB-null strain may have an unusually high propor-tion of ALCs. It is possible, as with LagC and CP2/cprB,that expression of rasD may continue to increase in thesubset of ubcB-null cells that form the slug, resulting ina higher, more extended expression pattern. However,rasD expression is not induced in the loose mass ofcells that remain at the base of the developing finger,and thus, the mechanisms by which UbcB proteincontrols spatial patterning are complex. The pheno-types observed in ubcB-null aggregates have not beenpreviously described in other mutant strains.

Analysis of chimeric organisms containing varyingratios of wild-type and ubcB-null cells in which thetwo strains are marked with lacZ reporters indicatesthat when there is a high enough proportion of wild-type cells, a small fraction of ubcB-null cells are able toform spores and participate in stalk formation. How-ever, it is clear that ubcB-null cells do not effectivelyparticipate in later morphogenesis, and in chimerascontaining predominantly mutant cells, the null cellsare found predominantly in the posterior/basal re-gions and do not participate in either stalk or sporeformation. The results are consistent with the inabilityof these cells to efficiently undergo the developmentaltransition that occurs at the time of cell-type differen-tiation. While ubcB-null cells are unable to efficientlyform a tip, this alone is not responsible for the ubcB-null phenotype; the addition of a small proportion ofwild-type cells leads to tip formation in chimeras butthis is unable to promote the proper development ofthe majority of the ubcB-null cells.

ubcB-Null Cells Show an Altered Pattern ofUbiquitin-containing ProteinsThe very high level of amino acid sequence identitybetween the UbcB ORF and UBCs from a variety oforganisms strongly suggests that UbcB encodes aUBC, which we have named UBC1. Our finding that

UbcB is an essential gene for progression past thefinger stage suggests that protein ubiquitination as anessential step in this process. This is consistent withour observations of an altered pattern of ubiquitinatedproteins in ubcB-null cells compared with wild-typecells. At least three ubiquitinated proteins exhibitchanges in their developmental pattern of stainingwith the antiubiquitin antibody on Western blots be-tween wild-type and ubcB-null cells. One major ubi-quitinated protein stains weakly in wild-type cellsduring early development, and then stains more in-tensely starting at the time of tip formation (12 h), thesame time that the ubcB-null phenotype is first ob-served. In ubcB-null cells, this band shows a moreconstant staining starting at the preaggregation stage(4 h). Another protein stains strongly through 12 h andthen disappears in wild-type cells. In ubcB-null cells,this band does not disappear, although the intensity ofstaining decreases. A third band exhibits a constant inwild-type cells from 4 h. In ubcB-null cells, this band issignificantly weaker in ubcB cells and this intensitydecreases even further starting at 12 h. It is of interestthat the observed changes in the developmental pat-tern of these three proteins between wild-type andubcB-null cells are different from each other. We do notknow if the changes in staining represent changes inthe level of ubiquitination, the rate of protein degra-dation, the kinetics of protein expression, or combina-tions of the three. Nor do we know whether either ofthese proteins are direct substrates of the UbcB proteinUBC1, whether the putative protein ubiquitination di-rected by UBC1 targets that protein for degradation bythe proteosome pathway, or whether the ubiquitina-tion functions in some other way. The timing of thechanges is consistent with UBP1 being required eitherdirectly or indirectly. Our data do not distinguishbetween changes in protein ubiquitination that are adirect function of UBP1 activity or pleiotrophicchanges that are indirectly the result of the ubcB-nullmutation. Nonetheless, these results indicate an effectof disruption of UbcB on protein ubiquitination thatcorrelates temporally with the ubcB-null cell pheno-type.

From the role of some UBCs in ubiquitin-mediatedprotein degradation, one possible explanation of thedevelopmental phenotypes exhibited by the ubcB-nullmutant is that the mutant cells are unable to degradeone or more proteins through the proteosome path-way whose turnover may be essential for develop-ment to proceed. This could either be the programmeddegradation of an endogenous inhibitor of develop-ment that regulates tip formation or a protein whosedegradation is required to activate a developmentalpathway. Ubiquitination has also been linked to othercellular functions, including protein targeting and cellfate decisions. In the case of the yeast MDR trans-porter Pdr5, yeast a-factor receptor, and mammalian

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growth hormone receptor, ubiquitination is involvedin regulating turnover via targeting of the protein tothe endosome (Egner and Kuchler, 1996; Hicke andRiezman, 1996; Strous et al., 1996). In Drosphila, the fatfacets gene, which encodes a deubiquitinating enzyme,is required for cell fate decisions in the eye (Jermynand Williams, 1995), opening up the possibility thatubiquitination is involved in mediating a diverse set ofcellular pathways.

At present, it is not known which ubiquitination-mediated pathway may be altered in ubcB-null cells,although the possibility that UBP1 may be involved intargeting specific proteins for degradation is consis-tent with previous results of mutations affecting aDictyostelium homolog of the human Tat-binding pro-tein, DdTBPa (Cao and Firtel, 1995). Members of theTBP family are also known to be components of the26S proteosome involved in protein degradation. TheDdTBPa gene appears to be essential for both vegeta-tive growth and multicellular development. Disrup-tion of one of the two copies of the gene results indevelopmental abnormalities that can first be ob-served at the finger stage. Expression of an antisenseconstruct in the partial knockout results in addi-tional morphologic abnormalities, while DdTBPaover-expression leads to a cytokinetic defect. Theseover-expressing cells also show abnormal morpho-genesis during the multicellular stages, with devel-opment arresting at the finger stage. These resultssuggest that protein degradation through the 26Sproteosome pathway(s) may be important for con-trolling specific stages during growth and develop-ment in Dictyostelium. Whether UBP1 and DdTBParegulate the same developmental processes orwhether either is involved in protein turnover is notknown.

A variety of proteins in addition to UbcB have beenshown to be required for cells to proceed normallypast the mound stage, form a finger, and continuethrough morphogenesis. These include: the transcrip-tion factor GBF; LagC, a putative cell surface signalingmolecule; TagB, a subunit of an MDR with a putativeserine protease domain; and the cAMP receptor cAR2(Saxe et al., 1993; Dynes et al., 1994; Schnitzler et al.,1995; Shaulsky et al., 1995). In addition, RasD has beenimplicated in tip formation (Reymond et al., 1986).Cells lacking myosin II and cells in which cytosoliccalcium has been depleted by expressing a consti-tutively active Ca21 membrane pump also arrest orare delayed at this stage (Springer et al., 1994; A.B.Cubitt, I. Reddy, J.G. McNally, and R.A. Firtel, un-published data). The diversity of the pathways thatmight be affected by these gene products suggeststhat multiple regulatory pathways are required tocontrol these transitions.

ACKNOWLEDGMENTS

We thank W. Loomis, A. Kuspa, and R. Hampton for helpful dis-cussions during the process of this work. The UbcB cDNAs weresequenced by the Dictyostelium sequencing core supported byUnited States Public Health Service Grant PO1HD30892. This workwas supported in part by United States Public Health Service grantsto R.A.F.

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