October

FEBS 10270 Volume 291, number 2, 222-224 0 1991 Federahon of European Biochemical Societies 00145793/91/$3 50 ADONIS

1991

0014579391009792

in Escherichia coli rpaH mutant defective in heat shock protein induction and inclusion

regation

AI. Gragerov, Instrture of

E.S. Martin, Molecuiat

MA.

Genettcs.

body formation

Krupenko,

USSR

Acadetnv

M.V Kashlev of Sctetms,

Moscow

and V.G. Niklforov 123182,

USSR

Wecetved 29 July 1991 Mutahons In the rpoH gene, encodmg u’*, an alterndtlve fdctor requued for transcnptlon of the heat shock genes, result m thecxtenslve aggregatmn of virtually all cellular protems and formatIon of mcluslon bodaes both under stress and non-stress conchhons Inhlbltors of protem synthesis suppress tlus aggregation, suggestmg that newly syntheszed proterns preferentially aggregate m rpoH mutants These data suggest that the heat shock protems are mvolved In acqulsmon of the soluble state (1 e correct conformation) of the bulk of mtracellular protems after their translation Heat shock protein, Inclusion body, Protcm folchng, rpolf mutant, Chaperone

1. INTRODUCTION The folding of proteins wIthun living cells has been considered for many years as a spontaneous process [l J Current studies recognized auxlhary protems called chaperones which are needed for protem foldmg, assembly and transport [2-4]. These protems belong to the heat shock proteins (hsps) and constitute at least two protem foldmg systems* hsp70, represented in E. cob by DnaK, and hsp60, represented by GroEL [4] These systems have been already Implicated m acquisition of active conformation of certain protems [5-S], m promoting folding and assembly of protems inserted mto mltochondria [lo-121, and m bmdmg of newly synthestzed polypeptlde chains [ 13,141 Nowever, It remains unclear how general their role IS are they mvolved m the folding of the maJority or only certain protems9 We address these questions usmg E. toll mutant m tpoH gene codtng for RNA polymerase 03* subunit responsible for the heat shock promoter recogmtton [ 15-181. These cells were defective m the mductlon of hsps We find that virtually all protems aggregate extensively dnd form inclusion bodies in rpoH mutant cells. 2. MATERIALS

AND METHODS

2 I Swarm artdpkasttrrds WC used Str‘uns SCl22, F-. Iaclarr~), trp(attt). plro(attt), mpC(r\). srrA. trra/(attt). and Kl65 SCl22, rpofff65 [19] Ptdsmld pRpoH IS

pDS2 [20] 2 2 Meclru cmd grow\III ~otrdtrrorr~ Strams wcrc grown In L-broth dt 30°C to the nud.logdnthmic

A I Grdgcrov. Inrtltutc of Molecular Gcncof Sc~enccs. Moscow I23 182, USSR

C’rrrrc~putt~k~~trcr addteu

tics, USSR hcadcmy

222

phase The cells were then mcubated at 42°C or dt 30°C for I h For plasnud-contammg cells amplcdhn (100 PgIml) was added to the mcchum Vlablhty of the cells after heat treatment was evaluated by platmg at 30°C 2 3 Preparatton atrd fk tronatron of cell Iysasares Cells were chilled on Ice, collected by centnfugatlon, and chsrupted by somcation (4-5 10-s bursts) on Ice m Laemmh loachng buffer [21] without SDS. contammg PMSF at the concentration 200 &ml The extent of cell dIsruptIon was momtored by phase contrast nucroscopy Usually less than 0 1% of cells remamcd Intact after somcation The cell lysates were centrifuged m Mrcrofuge I1 (Beckman) at I3 500 rpm for 2 mm SDS was dddcd to the supernatant The pellet was rmsed once with excess of the buffer, centrifuged and resuspended m SDScontammg loadrng buffer Before electrophoresls the samples were heated m a bollmg water bath for l-2 mm Equal volume of soluble (S) and msoluble (I) protem fractions were separdted on 10% SDSPAGE [2l] and Coomassle stamed 2 4 Mlcroscoprc rcclfnrqr~cs Cells were fixed for 30 min in 1% glutdrdldehydc, 0 1 M Nd-cacodyIdte, pH 7 3, dl ambient tcmperdture Flxatlon m 2% osrmum tetro. xlde. 0 2 M Nd-cacodylatc, pH 7 3 was for I2 h at 4°C Flxcd cells were treated with 0 5% uranyl acetate m maleate buffer, pH 5 2 for 90 mm at 4’C The bdctetId were then embcddcd m Epon Thm sectlons (less than 500 A} were stamcd with lead Citrate [22.23]

3 RESULTS E co/r strain K165 [ 191 carries an amber mutation m the rpoH gene, which encodes the heat shock sigma factor [16-181 and a temperature-scnsltive amber suppressor. At 30°C K165 grows normally despite the rcduccd amount of hsps [24, 251, but has lnnltcd ability to induce hsps syntheses. At 42°C the mutant cells are unable to form colomes, presumably due to cess&lon of hsr,s synthesis However, they contmuc to grow for at icast 1 h (as Judged by culture turbldlty) and rctam 100% viability durmg this tnne period. but fall to divldc (data not shown) WC incubated cells at 30°C or 42“C,

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Strsln

rpol-l

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Wl

PLssalcl

Temperature Frsetlon

30’ m-----7 s1sIsIsIsI

42”

3Q’

-52’

77

W.f.

l?g 1 Protem aggregation m ~poH165mutant at 42’C Cells were grown at 30°C and portions of the cultures were transferred to 4f0 for I h Soluble (S) and msoluble (I) fractions were obtamed as dcscrtbed m Materials and Methods and separated on 10% SDS-PAGE Note that msolublc fractions were loaded on gel m twice the amount of the corrcspondmg soluble fraction tpoH= Kl65 stram. wt = SC122 stram

disrupted them by sonic&on, and fractlonatcd the somcate mto soluble and insoluble fractions by brief centnfugatlon. Both fractions were analyzed by SDS- polyacrylamlde electrophoresls (Fig. 1) We found that rpoHi6.5 cells had much more proteins m the msoluble fraction than did the wild-type cells. Durmg incubation at 42°C the amount of protein m the pellet fraction of ~poH cells increased and reached 20-25% of total protein content after 1 h. Introduction of a plasmld carrymg a weld-type rpoH gene prevented protein aggregation in the rpoH mutant strain (Fig. 1) Phase contrast light microscopy revealed the appearance of refractive particles in rpoH165 cells after heating (data not shown) Electron microscopic examination of thm sectlons of rpoHi6S heat-treated cells showed that aggregated proteins formed typical mclusoon bodies (Fig 2), previously described for cells overproducmg certain mdivldual proteins [26] and for cells producing abnormal proteins m the presence of puromycm or ammo acid analogues [27] Thus OUI data show that normal proteins behave like abnormal proteins under conditions of hsps shortage No protein aggregation was observed when rpoH165 ceils were incubated at 42°C m the presence of antlblotic inhibitors of protein or RNA synthesis, while an antlblotlc mhlbltmg DNA synthesis did not prevent aggregation (Fig. 3). These data mdlcate that aggregates dre folmed predommantly by newly synthesized proteins. Pulse labeling showed that protem synthesis did occur m rpoH165 cells at 42°C at normal rates for at least 1 II. A substantial portion of the pulse-labeled proteins was msoluble (datd not shown). 4 DISCUSSION We observed cxtcnslvc agglegatton of protems leading to mcluslon body formation m ,poH tnutant cells dcfcctlvc m hsps synthesis Prcvlously inclusion body

-

-lP”

Fig 2 Electron mvzrographs of thm sectlons ofrpoHI64 and wild-type cells Cells were mcubdted for 1 11at 42°C and thm sections were prepared as described m Materials and Methods Selected mcluslon bodies are marked by arrows Wdd-type = SC122 top, rpoH165= Kl65. bottom

formation was described only for overproduced [26] or abnormal proteins synthesized m the presence of puromycm or ammo acid analogues [27] The most probable explanation of protcm aggregation m the rpoH mutant IS that some hsp(s) ale necessary for correct folding and/or assembly of the bulk of newly synthesized polypcptldes Conclusion that these ate newly synthesized proteins that predommantly aggregate m rpoH mutant was based on the observation that mhlbltlon of protein synthesis completely inhibited protem aggregation at the elevated temperature This suggests that most mature protems are not damaged ,it 42°C in tpoH mutants. Since m certain well-studied casts foldmg mtermedlates were shown to be much more temperature-sensitive and prone to aggrcgatlon than mature protems [28,29], one can speculate that these are folding mtermedlates of newly synthcslzed protcms which ,lre forming aggregates m ,poH cells. Protcrn dggrcgatlon m rpoH mutants was most pronounccd at the elcvatcd tcmperetulc of 42OC, but was 223

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Fig 3 Effect of rnhlbltors on the protem aggregation at 42°C m the r/~oHl65 mutant Cells were grown at 30°C and then shIfted to 42°C as described m Materlals and Methods with or wlthout addltlon of antlblotlcs NV = novoblocm (50 @ml), Sp = spectmomycm (50 ggImt), Cm = chloramphentcot (ZO&$ml), Rf = nfamplcm (SO,@nt), Tc = tetracyctme (20 pg/ml)

also readily detected at the non-stress temperature of 30°C It IS known that some protems retam solublhty when overproduced in the wild-type cells, It appears, however, that these protems (e g. P-galactosldase) hecame slgmficantly aggregated m rpoH mutants even at 30°C (our unpubhshed data and [30]) This shows that the proposed general function of hsps 1s required not only under stress, but also under normal condltrons, which is consistent with the mdlspensabllity of at least some hsps [3l-343 Which particular hsps protect proteins from aggregation m E colP The obvious candidates are GroE and DnaR proteins (see Introduction) To test this we are performmg experrments with rpoff mutants carrying plasmlds with groE or dnaK operon dc/tnow/e~gernenls We are grateful to E Bogddnova. D Cherny, C Gross, M Gottesmdn, S Lmdqulst dnd C Gntdndrls for dIscussIons and comments, and to C Gross, R Shakulov dnd S Smeokly for provldmg baCtCrldl strdlns dnd ptdsnxds

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