Ubiquitin gene expression in Dictyostelium is induced by heat and cold

shock, cadmium, and inhibitors of protein synthesis

A. MULLER-TAUBENBERGER, J. HAGMANN, A. NOEGEL and G. GERISCH*

Max-Planck-lnslitiil fiir Biochemie, D-8033 Martinsried bei Miinchen, Federal Republic of Gennany

* Author for correspondence

Summary

Ubiquitin is a highly conserved, multifunctionalprotein, which is implicated in the heat-shockresponse of eukaryotes. The differential ex-pression of the multiple ubiquitin genes inDictyostelium discoideum was investigatedunder various stress conditions. Growing D. dis-coideum cells express four major ubiquitin tran-scripts of sizes varying from 0-6 to 1-9 kb. Uponheat shock three additional ubiquitin mRNAs of0-9, 1-2 and 1-4 kb accumulate within 30min. The

same three transcripts are expressed in responseto cold shock or cadmium treatment. Inhibition ofprotein synthesis by cycloheximide leads to aparticularly strong accumulation of the largerubiquitin transcripts, which code for polyubiqui-tins. Possible mechanisms regulating the ex-pression of ubiquitin transcripts upon heat shockand other stresses are discussed.

Key words: ubiquitin, heat shock, inhibitors of proteinsynthesis, cadmium effects, Diclvosteliimi.

Introduction

Ubiquitin is a 76 amino acid protein, which is highlyconserved in all eukaryotes examined. Based on se-quencing either of the protein or of cDNA clones, theamino acid sequence of ubiquitin proved to be identicalin various insects (Gavilanes el al. 1982; Arribas et al.1986) and vertebrates (Schlesinger & Goldstein, 1975;Dworkin-Rastl et al. 1984; Wiborge/ al. 1985; Bond &Schlesinger, 1985). Compared to the human sequencethere are only two amino acid substitutions in Dictyo-stelium (Giorda & Ennis, 1987; Miiller-Taubenbergeret al. 1988) and three in yeast (Ozkaynak et al. 1984)and higher plant sequences (Vierstra et al. 1986;Gausing & Barkardottir, 1986). In most of the ubiqui-tin genes multiple coding units are aligned in tandemgiving rise to polyubiquitin precursors that need to beproteolytically cleaved in order to yield the functionalubiquitin monomers. In man (Lund et al. 1985),Dictyostelium (Westphal et al. 1986) and Saccharo-myces (Ozkaynak et al. 1987) certain ubiquitin geneshave been shown to encode fusion proteins in whichmonoubiquitin is extended at its carboxy terminus bysequences that are rich in basic amino acids. Thesesequences contain several cysteine residues in an ar-rangement that is reminiscent of the putative metal-binding sites of nucleic acid binding proteins. These

Journal of Cell Science 90, 51-58 (1988)Printed in Great Britain (G) The Company of Biologists Limited 1988

results suggest that the family of ubiquitin genesencodes at least two classes of precursor proteins,which differ in their destination and function withinthe cells.

The ATP-dependent non-lysosomal proteolysis thatis mediated by ubiquitin (Ciechanover et al. 1984;Hershko & Ciechanover, 1986) is most likely impli-cated in the heat-shock response (Finley et al. 1984;Parage/o/. 1987). In chicken embryo fibroblasts (Bond& Schlesinger, 1985) and yeast (Finley et al. 1987)ubiquitin itself is a heat-shock-induced protein. Thishas not been observed in Dmsophila (Arribas et al.1986) or Dictyostelium (Giorda & Ennis, 1987). Otherfunctions of ubiquitin are suggested by its location inthe nucleus and on the cell surface. Ubiquitin iscovalently linked to histones II2A and I12B (Gold-knopf et al. 1975), preferentially in regions of activelytranscribed chromatin (Matsui et al. 1979). The lym-phocyte homing receptor (Siegelman et al. 1986) andthe platelet-derived growth factor (Yarden et al. 1986)are cell-surface proteins reportedly ubiquitinatcd.

Dictyostelium discoideum is a eukaryotic micro-organism in which the expression of specific genes islinked to discrete steps of development. Upon star-vation of growing cells, development proceeds througha pre-aggregation phase to cell aggregation, which is

51

followed by cell-type specification and terminal differ-entiation within a multicellular body. Among thecDNA clones that we have used to identify mRNAspecies that are expressed during early development(Gerisch et al. 1985), there was one that hybridized tomultiple mRNA species. This clone proved to encodeubiquitin. Using this clone, a multigene family en-coding seven ubiquitin mRNA species with differentsizes could be demonstrated (Westphal et al. 1986).Larger mRNA species have been shown to representpolyubiquitin messages (Giorda & Ennis, 1987). Allthe larger mRNAs were found to accumulate duringearly development while the small ones declined (West-phal et al. 1986). In the present paper we show thatheat shock and other stresses cause accumulation ofubiquitin mRNAs, and that these mRNAs are dramati-cally overexpressed in cells treated with cycloheximideor other inhibitors of protein synthesis.

Materials and methods

Cell culture and developmentCells of Dictyosteliuni discoideum strain AX2-214 weregrown axenically at 21 °C as described (Malchow et al. 1972).To initiate development, cells were harvested at densities of2-4X 10°cells ml""1, washed and cultivated in suspension on agyratory shaker at 150 rev. min" in 17 mM-Soerensen phos-phate buffer at a cell density of 1 X 107 per ml.

Heat and cold shock and heavy metal treatmentTemperature shocks and heavy metal ions were applied in thepresence of nutrient medium to cell cultures grown up todensities of 1-4X 106 cells ml" ' . The cells were heat shockedby shifting the temperature from 21 °C to 30°C. For recoverythe temperature was shifted back to 21CC. The cells were coldshocked by shifting the temperature from 21 °C to 4°C andkept in the nutrient medium under shaking conditions at100 rev. min~'.

Inhibition of protein synthesisInhibitors of protein synthesis were added either to growingcells in the presence of nutrient medium as described for heatshock (see Fig. 5), or to cells starved in phosphate buffer asfor the initiation of development (see Figs 4, 6). Cyclohexi-mide and anisomycin were purchased from Sigma, emetinefrom Serva (Heidelberg). The rate of protein synthesis wasdetermined by adding [3sS]-L-methionine (Amersham,370 kBq per 1x10° cells) at 30 min after the addition ofinhibitors and to control cells. Incorporation of the 35S-labelwas stopped after 10 min of incubation by the addition oftrichloroacetic acid, final concentration 10%. The pelletswere washed twice with trichloroacetic acid and the radioac-tivity in pellets obtained after zero time of incorporation wassubtracted as background from the precipitable radioactivity.[3SS]Methionine incorporation into untreated control cellswas set as 100%.

RNA isolation, Northern blotting and hybridizationFor Northern blots total cellular RNA was purified byphenol-chloroform extraction, separated by gel electrophor-esis in 1-2% (Figs 3, 4, 6) or 1-5 % (Figs 1, 2, 5) agarose inthe presence of 6 % formaldehyde (Maniatis et al. 1982), andtransferred to nitrocellulose (BA85, Schleicher and Schuell).A 10-jUg sample of RNA was applied per lane. The blots werehybridized for 16-18 h with the nick-translated ubiquitin-specific cDNA fragment UB1U (Westphal et al. 1986) at37°C in 50% formamide, 2XSSC ( lxSSC is 0-15M-NaCl,15mM-sodium citrate), 4xDenhardt's, 1 % Sarcosyl, 0-12M-sodium phosphate buffer, pH 6 8 , and 0-1% SDS. RNAsizes were determined using an RNA ladder (BethesdaResearch Laboratories) as marker. For comparison, either acloned 1 -5-kb EcoVA cDNA fragment from the coding regionof theD. discoideum 120K (103.<l/r) gelation factor, or a 12-kb £coRI fragment from the coding region of Q'-actinin wasused (Whkcetal. 1986).

Results

Differential expression of ubiquitin genes duringtemperature shocks and cadmium treatmentIn growing D. discoideum cells four major ubiquitintranscripts of 0-6, 0-7, 1-5 and l-9kb and one minortranscript of 1-4 kb were expressed. In cells starved for5h ubiquitin transcripts of 0-9 and 1 -2 kb appeared,and the amounts of the 1-4-, 1-5- and L9-kb transcriptsincreased (Fig. 1, first and fourth lane). By shiftinggrowing cells from 21 °C to 30°C, a temperature thatinduces maximal heat-shock responses in D. discoid-eum (Loomis & Wheeler, 1980), the accumulation ofthe same ubiquitin transcripts as during starvation wasinduced or-enhanced (Fig. 1, second lane). No sub-stantial heat-shock effect was observed for the 0-6- and0-7-kb ubiquitin transcripts.

Since the effects of heat shock in Drosophila andmammalian cells are mimicked by other stresses includ-ing exposure to heavy metals (Ashburner & Bonner,1979; Ananthane/ al. 1986; Burdon, 1986), we testedthe influence of cadmium, zinc and cobalt on ubiquitingene expression in D. discoideum cells. Treatment with100,itM-Cd(NO3)2 for 30min induced, similar to heatshock, the 0-9- and L2-kb transcripts and stronglyenhanced accumulation of the 1-4-, L5- and L9-kbspecies (Fig. 1, third lane). No effect was seen underthe same conditions with 1 mM-ZnSC^ or 0-8 mM-CoCl2.

The heat-shock effect on ubiquitin gene expressionwas fast; accumulation of the induced mRNAs beganwithin 15 min and reached a maximum within 30 min(Fig. 2A). After shifting the cells back to 21 °C, theyreturned within 30 min to about the initial state ofmRNA expression (Fig. 2A). After 1 and 2h of recov-ery at 21 °C the cells expressed even less of the heat-shock-induced 1-5- and 1-9-kbmRNA than they hadexpressed before the heat shock (zero time in Fig. 2A).

52 A. Miiller-Taubenberger et al.

Oh 5h

Hs Cdkb

1-9-

1-5-1 -4 -1 - 2 -

0 - 9 -iff!m

0 - 7 -0 - 6 -•!••

Heat shock Recovery

15 30 60 ' 30 60 120 Min

0-9

0-7

0-6

i / :

Fig. 1. Ubiquitin transcript accumulation after heat shock,cadmium treatment and initiation of development bystarvation. RNA isolated from growth phase cells of D.discoideum was extracted at the same time from controlcells kept in nutrient medium at 21 °C (C at Oh), from cellsincubated for 30min at 30°C (Hs), and from cells treatedfor 30min with 100fiM-Cd2+ at 21 °C (Cd). Alternatively,cells were starved in phosphate buffer and harvested after5 h of development at 21CC (C at 5 h). Northern blots werelabelled with the ubiquitin-specific cDNA probe UB1U.

Microscopic examination indicated that the cellsremained intact, excluding the possibility that thelower expression of these mRNAs was due to lysis.Furthermore, as an internal control, the expression ofthe mRNA for a-actinin was used. This mRNAencodes a cytoskeletal protein that is known to bepresent in all stages of growth and development (Witkeet at. 1986). No significant change in the amount of thisRNA was found during the heat shock or within therecovery period (Fig. 2B).

When growing cells were shifted from 21 °C to 4°C,the same ubiquitin transcripts accumulated as in heat-shocked cells (Fig. 3). The accumulation was slowerand less extensive during the cold shock than after aheat shock. The cold-shock response is not a uniquefeature of polyubiquitin genes; a similar accumulationupon heat and cold shock has been observed foranother developmentally regulated transcript of D.discoideum (Maniak & Nellen, 1988).

B

Fig. 2. Ubiquitin transcript regulation during recoveryfrom heat shock. Growth-phase cells were incubated at30°C for the indicated times (heat shock) and shifted backafter 60 min to the normal growth temperature of 21 °C(recovery). Northern blots were either labelled with theUB1U ubiquitin-specific probe (A), or with an a'-actinin-specific cDNA probe (B).

Overexpression of ubiquitin transcripts by theinhibition of protein synthesis

The inhibition of protein synthesis was tested as apotential stress factor and was found to enhancestrongly ubiquitin transcript accumulation. Starvedcells were incubated with three different concen-trations of cycloheximide to compare accumulation ofubiquitin transcripts with the inhibition of proteinsynthesis. After 2h of incubation a substantial increasein the amounts of transcripts was observed at allcycloheximide concentrations tested (Fig. 4). At thelowest concentration, 50jUgml~', [3;>S]methionine in-corporation was reduced by 68% and accumulation ofubiquitin mRNAs was clearly detectable. Thisincreased accumulation was specific for the five larger

Ubiquitin gene regulation 53

0 15 30 60 120 Min 0 50 250 500 gCHX

Fig. 3. Ubiquitin transcript accumulation in response tocold shock. Growth phase cells were shifted from 21 °C to4°C and incubated at this temperature for the timesindicated. Northern blots were labelled with the ubiquitin-specific UB1U cDNA.

ubiquitin transcripts. These mRNAs were maximallyexpressed with 250/.tgml~' cycloheximide, which in-hibited [35S]methionine incorporation by 90%. Thesame dramatic accumulation of ubiquitin mRNAs wasobserved with 500/igmP' cycloheximide, a concen-tration that caused 98% inhibition of [ SJmethionineincorporation (Fig. 4).

Other elongation inhibitors of protein synthesisshowed the same pattern of ubiquitin mRNA induction(Fig. 5): 40ftM-anisomycin and 500,iiM-emetine in-hibited [35S]methionine incorporation by 84% and77%, respectively, and showed the same or slightlyweaker ubiquitin mRNA accumulation as SO^gmP1

cycloheximide which inhibited incorporation by 68 %.

Accumulation of the ubiquitin transcripts was notdue to a general stabilization of mRNA by cyclohexi-mide (Kelly et al. 1987). The amounts of ubiquitinmRNAs remained high for at least 6h in the presenceof cycloheximide (Fig. 6A), but a reference mRNAencoding another protein disappeared in the presenceof cycloheximide (Fig. 6B). The mRNA used here as areference encoded a cytoskeletal protein, the 120Kgelation factor (Condeelise/ al. 1982), and was presentthroughout growth and development in untreated cells(A. Noegel and M. Schleicher, unpublished results).

kb

1-9 —

1-5 —1-4 —1-2 —

0-9 —

0-6 —

100 32 10 2 %

Fig. 4. Ubiquitin transcript accumulation at reduced levelsof protein synthesis. Cycloheximide (CHX) was added atthe concentrations indicated immediately after onset ofstarvation of growth phase cells in phosphate buffer. RNAwas extracted after 2h of starvation and hybridized inNorthern blots with the ubiquitin-specific UB1U cDNA.The rates of protein synthesis indicated at the bottom weredetermined by [35S]methionine incorporation as describedin Materials and methods. Compared to the previousfigures, the exposure time of the autoradiogram wasreduced in order to account for the over-expression ofubiquitin transcripts at high cycloheximide concentrations.

Discussion

Ubiquitin as a putative mediator of stress responsesUbiquitin genes have previously been shown in yeast(Finley et al. 1987) and chicken (Bond & Schlesinger,1985) to be expressed in response to heat shock. Theresults obtained with D. dtscoideum show that thiseffect is more general. Evidence for a function ofubiquitin in protecting cells against heat shock isprovided by the increased sensitivity against heat shockof a yeast mutant in which a gene encoding polyubiqui-tin is disrupted (Finley et al. 1987). Strong expressionof heat-shock proteins at non-heat-shock temperaturein the ts 85 mouse cell line, which carries a mutationresponsible for thermolability of the ubiquitin-acti-vating enzyme El, suggests that the El-ubiquitincomplex is involved in the suppression of heat-shockgenes at normal temperatures (Finley et al. 1984).

Denatured proteins have been shown to initiate heat-shock responses (Ananthan et al. 1986) and it has beensupposed that it is the ubiquitin system that connectsprotein denaturation to the induction of heat-shock

14 A. Muller-Taubenberger et al.

CHX AN EM

kb

1-9 —

1 - 5 -1 - 4 -

1-2 —

0 - 9 -

lit0-7 —

0 - 6 -»• • *100 32 16 23%

Fig. 5. Comparison of cycloheximide, anisomycin andemetine effects on ubiquitin transcript accumulation.Growth phase cells were treated for 30min in the presenceof nutrient medium with 50|Ugml~' of cycloheximide(CHX), 40^M-anisomycin (AN), or 500f(M-emetine (EM).The rate of protein synthesis as determined by[3SS]methionine incorporation in the inhibitor-treated cellsis given as a percentage of synthesis in control cells (C).

genes (Finley et al. 1984; Munro & Pelham, 1985). Theprincipal assumption is that denatured proteins com-pete with a heat-shock regulator protein for El-acti-vated ubiquitin, such that in the presence of denaturedproteins more of the regulator protein exists in a non-ubiquitinated state. The regulator protein is thought tobe inactive when ubiquitinated and to activate the heat-shock promoters in its free state. Relevant regulatorproteins have been designated as heat-shock activatorprotein or heat-shock transcription factor HSTF(Zimarino & Wu, 1987).

lleat-shock-induced polyubiquitin mRNAaccumulation

The results presented in this paper indicate thatexpression of a group of ubiquitin transcripts isincreased in response to heat or cold shock, to Cd2+

treatment, or to induction of normal development bystarvation. Even higher levels of expression wereobtained with cycloheximide or other inhibitors ofprotein synthesis. The effect of all these treatments isrestricted to the larger ubiquitin transcripts, whichprobably all encode tandem repeats of ubiquitin se-quences (Giorda & Ennis, 1987). Transcripts of 0 6and 0-7 kb, which were not regulated by these treat-ments, are too small to code for polyubiquitins. The

0-6-kb transcript encodes a mono-ubiquitin that islinked at its C-terminal end to a basic polypeptide(Westphal et al. 1986). The 0-7-kb transcript may codefor a mono-ubiquitin with another C-terminal sequenceor for a di-ubiquitin. These results indicate peculiarregulatory mechanisms for the polyubiquitin tran-scripts and suggest functions for the C-terminallyextended mono-ubiquitin that are not involved in stressresponses.

The parallel effects of heat shock or other stressesand of the initiation of Dictyostelium development bystarvation (Westphal et al. 1986) suggest common stepsin the pathways of ubiquitin gene expression. More-over, polyubiquitin genes are not the only genes thatare affected by heat shock and the initiation of develop-ment. A set of repeated sequences, which comprise anelement homologous to the heat-shock promoter inDrosophila, is induced in D. discoideum both by heatshock and during induction of development by star-vation (Zuker et al. 1983). Despite these similarities itis unlikely that development and the responses tovarious stresses including cold shock are all initiated bythe same alterations within the cells.

The cycloheximide effect and its bearing on theregulation of polyubiquitin genes

The results shown in Fig. 6 together with those ofFig. 4 indicate that the amounts of polyubiquitinmRNAs continue to increase for several hours withnegligible de novo synthesis of proteins. If activation ofthe ubiquitin genes is mediated by heat-shock activatorprotein, as discussed above, this protein should beeither long-lived or continuously delivered from a largestore or precursor pool. This conclusion is consistentwith the results of Zimarino & Wu (1987), whichindicate a fast reversible conversion of the activatorprotein from an inactive to an active form without arequirement for protein synthesis.

It has been suggested that cycloheximide indiscrimi-nately stabilizes mRNAs in D. discoideum (Kelly et al.1987). However, decay of the mRNA of a cytoskeletalprotein, the 120K gelation factor, showed that mRNAscan be degraded in the presence of cycloheximide(Fig. 6B). Thus, accumulation in the presence ofcycloheximide is not a general feature of D. discoideummRNA. It is likely that cycloheximide affects thestability of polyubiquitin mRNA as well as the rate oftranscription.

It is possible that cycloheximide stimulates ubiquitingene expression by producing incomplete polypeptidesthat are readily ubiquitinated because they do not foldcorrectly. But if this were the only way in whichcycloheximide causes ubiquitin gene expression, amaximal effect should be observed at intermediatecycloheximide concentrations that only partially inhibitprotein synthesis. However, at concentrations that

Ubiquitin gene regulation 55

2h 4h

0 250 0 250 0 250 figml"1

CHXkb

1-9 —

3 - 2 -

B

inhibited protein synthesis almost completely, poly-ubiquitin transcripts were maximally expressed

(F'R- 4).Another mechanism of cycloheximide induction of

polyubiquitin genes is suggested by the dual role ofubiquitin as a hcat-shock-induced protein and a media-tor of heat-shock responses. Therefore an autoregu-latory cycle may exist in which a heat-shock activatorprotein would play a key role in negative feedbackcontrol of polyubiquitin genes (Finley el al. 1984;Munro & Pelham, 1985). On the basis of similar resultsin Drosophila, a loop for heat-shock proteins has beensuggested with I ISP 70 as a self-regulating protein(DiDomenico el al. 1982). Two results obtained withD. (liscoideiiM support negative feedback regulation ofubiquitin. First, during recovery of cells from a heatshock, an undershoot of ubiquitin transcripts is ob-served (Fig. 2). This result suggests inactivation ofubiquitin genes by the high concentrations of ubiquitinthat result from ubiquitin overproduction during theheat shock. Second, the extraordinarily strong ex-pression of ubiquitin genes that is caused by cyclohexi-mide (Figs 4—6) might be a consequence of disruptinga negative feedback cycle in which ubiquitin is in-volved. If ubiquitin is continuously consumed and no

Fig. 6. Time course of ubiquitintranscript accumulation in the presenceof cycloheximide (A) compared to thedegradation of a reference mRNA (B).Cycloheximide (CHX) was added toone sample of cells immediately afteronset of starvation in phosphate buffer,another sample was run in parallel as anuntreated control. RNA was extractedafter 2, 4, and 6h of starvation.Northern blots were either labelled withthe UB1U cDNA for ubiquitintranscripts (A), or with a cDNA probefor mRNA of the 120K gelation factorthat served as a reference (B).

longer produced when protein synthesis is blocked, theubiquitin genes would become fully activated and theuntranslated transcripts would accumulate.

We are grateful to Mrs B. Book for organizing themanuscript. For critical reading of the manuscript we thankDr J. Segall, and Dr R. Kulka, Hebrew University, forstimulating discussions. A. Miiller-Taubenberger gratefullyacknowledges a Kekule-Stipendium of the Fonds derChemischen Industrie.

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