Early intermediates in bacteriophage lambda prohead assembly

VIROLOGY

96, 341-367 (1979)

Early Intermediates

in Bacteriophage

Lambda

Prohead

Assembly

HELIOS MURIALDO Department

of Medical

Genetics, University

of Toronto,

Toronto,

Canada, M5S lA8

Accepted March 2, 1979

The morphogenesis of phage h proheads is under control of the four phage genes B, C, E, and Nu3 and the host cell gene groE. To determine if the assembly of the prohead proceeds via the formation of subassembly complexes, the proteins synthesized in E. coli following infection with h phage were analyzed by analytical sedimentation in glycerol gradients. The phageinduced proteins were labeled in viva by incorporation of V-labeled Met, and the protein composition of the fractions of the gradients was determined by electrophoresis in polyacrylamide gels followed by autoradiography. It was found that gpB sediments at positions corresponding to 25 S and 30 S, and that two polypeptides, derived from gpC, sediment as 30 S. The 25 S and 30 S complexes accumulate in a--infected cells, but no complexes are formed in A.E-B--infected cells. The formation of the 30 S complex, but not of the 25 S complex, is blocked in hE-C--infected cells. A host-controlled band cosediments with gpB in the 25 S position, but not in the 30 S position. Using a recombinant A phage carrying the host groE gene, the band cosedimenting with gpB was identified as the product of the groE gene. The groE gene product and gpB seem to form a complex since amber peptides of gpB (about % the size of the wild-type product) still cosediment with gpgroE in the 25 S position of the gradient. The formation of the 30 S complex is blocked, and the synthesis of the 25 S complex strongly inhibited, in groE missense mutant cells infected with A,?-. The results suggest that gpB and gpgroE interact at an early stage in prohead morphogenesis to form a 25 S complex which seems to be a precursor of a 30 S gpB- and gpC-containing complex. The existence of a gpB-gpC complex suggests that these two gene products interact directly. Since gpB is located in the head-tail junction of the phage, it seems highly likely that the polypeptides pX1 and pX2, derived from gpC, are also located at the head-tail junction. INTRODUCTION

The morphogenesis of the head of bacteriophage A is controlled by ten phage-coded genes and atleast one host gene (Campbell, 1961; Parkinson, 1968; Boklage et al., 1973; Jara and Murialdo, 1975; Murialdo and Siminovitch, 197213;Georgopoulos et al., 19’73; Sternberg, 1973). The process proceeds first by the formation of an intermediate called the prohead, an icosahedral structure containing no DNA (Hohn and Hohn, 1974; Hohn et al., 1974; Kaiser et al., 1974, 1975). Replicating concatemeric A DNA is then cut and packaged into proheads (Hohn and Hohn, 1974; Hohn et al., 1974; Kaiser et al., 1975; Becker and Gold, 1975; Becker et al., 1977a). Finally, the DNA-filled head structures are modified at one corner so that tails can attach and complete the virion

(Casjens et al., 1972; Casjens, 1974). Each of these phases is under the control of a specific set of gene products. The genes specifically involved in the assembly of proheads are B, C, Nu..Y,and E of the phage, and the host gene. groE. The remaining genes control DNA packaging and head completion. Observations on thin sections of X-infected cells suggest that a core-containing prohead is the precursor of the empty prohead which, in turn, is the precursor of the mature, DNA-filled head (Zachary et al., 1976). Little is known, however, about the earliest steps in the formation of the corecontaining prohead, or the molecular makeup of the precursors that enter its assembly. The construction of the prohead proceeds through a complex set of reactions in which certain structural components of the finished shell arise as a result of alterations of the

341

0042~6822/79/100341-27$02.00/O Copyright All rights

0 19’79 by Academic Press, Inc. of reproduction in my form reserved.

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MURIALDO

primary gene products. The prohead is composed of about 420 molecules of gpE arranged in the form of an icosahedral shell (Buchwald et al., 1970a; Casjens et al., 1970; Huntley and Kemp, 1971; Casjens and Her&ix, 1974;Williams and Richards, 1974). There are also four species of minor proteins: about 4 molecules of gpB and about 8 molecules of gpB*, the cleaved derivative of gpB, and 5 to 6 molecules each of pX1 and pX2 (Hendrix and Casjens, 1974a; 1975; Hohn et al., 1975). These last two protein species arise by fusion of gpC to a small fraction of the total gpE molecules and subsequent cleavage of both the gpE- and the gpC-derived moieties of the fusion product. The difference between pX1 and pX2 lies in the nature of the fused gpC derived fragment, which is shorter in pX2 than in pX1 (Hendrix and Casjens, 1974a, 1975; Hohn et al., 1975; Ray and Murialdo, 1975; Murialdo and Siminovitch, 1972a). We have previously summarized the indirect evidence that the minor components of the capsid, gpB, gpB*, and pX1 and pX2 are the structural components of the headtail junction and have presented a model for the molecular arrangement of these subunits (Murialdo and Ray, 1975). Direct evidence for the involvement of gpB and of gpB* as structural components of the head-tail junction has been obtained recently (Ray and Murialdo, unpublished; Tsui and Hendrix, 1978). Formally, there are two possible ways for the incorporation of a connector in the prohead. The connector could either be added to a preexisting icosahedral shell by the proper modification of one corner, or it could be made flrst and the capsid membrane subsequently assembled upon it. According to the latter model of prohead assembly, the connector might be viewed as a nucleus for the polymerization of the major membrane protein, gpE (Murialdo and Becker, 1978b). In earlier studies we showed that extracts of AE--infected cells contain all the ingredients required to nucleate the assembly of biologically active proheads when a source of gpE protomers is added (Murialdo and Becker, 1978a).Thus, the E- extract seemed the logical place to search for the appropriate

phage and host-derived assembly precursors. This paper describes such a search, using as the main tools of analysis the fractionation of cellular components by sedimentation, and the identification of specific proteins by electrophoresis in SDS-containing polyacrylamide gels. MATERIALS

AND METHODS

(a) Phuges Lambda bZcIsgg,Ab221CZ26, ~am915b221c~261 ACam73&2d26, ~am815b221cZ26, m&am,,bzz1cIz6,and hEama,JVtiam.,b,,,cI,, have already been described (Ray and Murialdo, 1975). hCam,3,Eamslsbzz,cZ26 was constructed by a cross between hCam,,+Z,, (Parkinson, 1968) and AEam,,,b,,,cZ,,, using TC600 (supE> as a host. The lysate of the cross was plated on the same cell strain and clear plaques that did not grow when stabbed to a lawn of QD5003 (supF) were selected for further characterization. (Phages carrying the Cam,,, mutation do not grow on strains with a supF suppressor.) Two isolates that were Cam,,, and Earn,,, when tested by marker rescue on a series of 594 lysogens carrying prophages with mutations in late genes (Murialdo and Becker, 1978a) were also tested for resistance to inactivation by EDTA (Parkinson and Huskey, 1971). They proved to be as resistant as the AEamelsb221c126 parent. For the construction of ABam,Eam,b,,,cI,,, ABam,Eam,cI,,,Sam, (described by Murialdo and Siminovitch, 1972b) was crossed with Ab221cZ26, and since the Sam, phage does not plate on supE strains, the lysate of the cross was plated on TC600 at 32”. Clear plaques were checked for the presence of the amber mutations Barn, and Earn+ and the presence of the b,,, deletion was detected by the resistance to EDTA, as already described. The DNA of Agti contains only one site susceptible to attack by R.EcoRl endonuclease, the one between the EcoRl fragments A and C. Fragment B of its DNA is missing altogether. The DNA of AgtavaBAD consists of a fragment of E. coli DNA carrying the araB, A, and D genes inserted into a deleted ADNA. The AgturaBAD molecule has two R.EcoRl sites, one between EcoRl

LAMBDA

PROHEAD

ASSEMBLY

343

total lysis, DNase was added to a concentration of 1 pg/ml, and the lysates were incubated 30 min at 37”. Phage titers were around 2-4 x 10lO/ml. The lysates were cleared by centrifugation at 9000 g for 20 min and the phage in the supernatant was pelleted by centrifugation at 7500 rpm in the GS-3 rotor of a Sorvall centrifuge for 16 hr at 4”. The pellet was resuspended in 8 ml of dilution buffer, then held overnight at 4” without shaking, and the remaining cell debris was removed by low-speed centrifugation. The phages were subsequently (b) Bacteria banded in a CsCl density gradient. The band The E. coli K12 strains used were: 159, was collected by lateral puncture with a uvrA-, sup”; 594, SmR, sup”; TC600, suII+ syringe, mixed with a CsCl solution with a (supE); and QD5003, suIII+ (au@), all from density of 1.5 g/cm3, and banded again. After our collection (Murialdo and Siminovitch, this second banding the phage was dialysed 19’72a). Strains groEA140 and groEB764 extensively against dilution buffer. (Georgopoulos et al., 1973) were a gift from A. D. Kaiser, whereas strain groEA97 was obtained from R. W. Hendrix. E. coli 594 (fl DNA Preparation (AAam,,~am,~Z,,~am,) and 594(Mam,,Phage preparations of @-ti and AgtaraBAD Bam,,,cZ,,,Sam,) were used for the in vitro were diluted to 5 OD,,, units/ml (about DNA packaging assay. They are from our 2 x 1012phages/ml) and the DNA was excollection (Murialdo and Becker, 1977). tracted by gentle rolling with freshly distilled phenol saturated with 0.5 M Tris, 0.05 M EDTA, pH 9.0. The extraction (c) Media procedure was repeated once more and the The composition of L broth, nutrient aqueous phase was dialyzed against two broth, dilution buffer, RM medium, RM-Mg changes of 600 vol of a buffer containing medium, and solid medium used for bacterial 0.11M NaCl, 0.02 M Tris-HCl, pH 7.6, and growth and for plaque assays has been 0.1 mM Na, EDTA. DNA from E. coli K12 described earlier (Murialdo and Siminovitch, strain TC600 was a gift of M. Gold. The 1971). procedures for preparation of this DNA were similar to those used for the preparation (d) General Techniques of phage DNA except that several extracThe methods for preparing and plating tions with phenol were carried out instead cells and phage stocks have already been of just two. described (Buchwald et al., 1970a; Murialdo and Siminovitch, 1972a). Phage crosses (g) Enzymes and Special Chemicals were carried out according to the method Pancreatic deoxyribonuclease (DNase I) described by Parkinson (1968). and pancreatic ribonuclease (RNase A) were purchased from Worthington Biochemical (e) Phage Lysates for DNA Preparation Corp. Endodeoxyribonuclease EcoRl was One liter of lysate was prepared by in- obtained from Bio Labs, and polynucleotide fecting cells, 594, at a density of about 1 synthetase (ATP) (DNA ligase) from T4x 107/ml with phages at a multiplicity of infected cells was obtained from Miles about 0.2. After 6-7 hr of incubation at 38” Laboratories. Bovine serum albumin, fracwith vigorous aeration, lysis ensued. Two tion V, and agarose type II were from Sigma milliliters of chloroform was added to ensure Chemical Co. The acrylamide was purchased AA fragment and the E. coli DNA, and the second one between the E. coli DNA and EcoRl AD fragment (R. W. Davis, unpublished, Cold Spring Harbor Course on “Advanced Bacterial Genetics,” 1979). These two phages were provided by D. A. Spandidos. For nomenclature on the R.EcoRl restriction sites and fragments of A DNA, see Thomas et al. (1974) and Murray and Murray (1974). Phage T5 was obtained from M. L. Pearson,

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MURIALDO

from Eastman Kodak Co., and the sodium dodecyl sulfate “especially pure” from British Drug House. These two latter products were used without further purification. The polypeptide Trasylol (aprotinin), which is a proteinase inhibitor (Briseid, 1970), was purchased from Boehringer Ingelheim Ltd. Phenylmethyl-sulfonylfluoride (PMSF), a serine protease inhibitor, was purchased from New England Nuclear. Polyethylene glycol (Carbowax 6000) was from Union Carbide; Brij-58, a nonionic detergent, was obtained from Atlas Chemical Co.; and Spectrafluor butyl-PBD was from Amersham/Searle. Other chemicals used were the purest grade commercially available. (h) Construction In Vitro

of Hybrid DNA Molecules

Each DNA was digested with EcoRl restriction endonuclease in a reaction mixture (0.115 ml) containing 0.075 M Tris-HCl buffer, pH 7.4, 0.01 M MgS04, 70 pg/ml of DNA, and 5 units (as defined by the manufacturer) of enzyme. The digestion was carried out at 37” for 80 min. Judging from the band pattern of the DNA fragments in agarose gel electrophoresis, hgti DNA is totally digested and E. coli DNA is extensively, if not totally, digested under these conditions. At the end of the incubation period the reaction was stopped by the addition of 0.5 M EDTA, pH 8.0, to a final concentration of 0.02 M. After a Smin incubation at 65”, the reaction mixtures containing the phage and E. coli DNA were mixed in equal proportions, 5 M NaCl was added to a final concentration of 0.3 M, and the DNA was precipitated with 3 vol of cold, 95% ethanol at -15” for 15 hr. After centrifugation at 17,000 g for 20 min at 4”, the supernatant was removed and the pellet was washed with cold 70% ethanol. The precipitate was dried in a vacuum desiccator at 4” and then dissolved in 0.25 ml of buffer containing 0.1 M Tris-HCl, pH 7.0, 0.05 M NaCl, and 0.1 mM EDTA. The conditions described by Murray and Murray (1974) were used for ligation, except that the incubation was for 18 hr at 9“. These experiments were com-

pleted in 1976, before the Canadian MRC guidelines for the handling of recombinant DNA molecules came into effect. Nevertheless, the equivalent of MRC level A containment was used for these experiments. (i) I;FY

Packaging

g LE. Coli Hybrid .:

The system described by Murialdo and Becker (1977) was used for in vitro packaging. A partially purified fraction of gpA and a sonic extract of an induced culture of 594(~am.,~am,,cI,,~am,) were used for the first stage of the reaction. The second stage was carried out using an extract of preinduced 594()IAam,,Bam,~Z,,,Sam,) pared by the lysozyme freeze-thaw method of Kaiser and Masuda (1973). Phage titers obtained varied with the efficiency of the assay. Usually about 2 x 105-lo6 PFU/pg of DNA were obtained when the phages assembled in vitro were plated on 594. (j) Electrophoresis

of DNA

Electrophoresis of the DNA in agarose gels was performed in a horizontal slab apparatus using the borate-containing buffer system of Mizuuchi and Nash (1976). Agarose concentration was usually 0.8%. Before electrophoresis the samples containing DNA were diluted in borate buffer mixed with %o the volume of a solution containing 100 m&f EDTA, 5% sodium dodecyl sarcosinate, 0.02% bromphenol blue, and 50% glycerol. The mixtures were heated for 5 min at 70”, cooled rapidly on ice, and loaded onto the gels. Voltage, time of electrophoresis, and staining procedures were those of Mizuuchi and Nash (1976). (k) Selection of XgroE Transducers

After ligation, the DNA was packaged into X proheads and resulted in the generation of phages at a concentration of 2 x 105106/ml, when plated on 594. AgroE-transducing phages were selected by plating the phage stocks which arose after packaging of ligated DNA on groEB764 cells. Plaques appeared at a frequency

LAMBDA

PROHEAD

of 10V3 whether hgti or AgtaraBAD was used as the transducing A vector. The plaques were purified by two consecutive clonings and small stock lysates were prepared for characterization. In order to be certain that the phages which arose after plating the transductants on a groEA or groEB host were not Ac mutants (Georgopoulos et al., 1973), we compared the properties of known AEmutants with those of the putative AgroE-transducing phages. The epsilon phages arose at a frequency of lo-‘, whereas AgroE phages were found at a frequency of lo- 3. In addition, the AE phages were specific for the type of groE host on which they were originally selected, i.e., AEA phage selected on a groEA host plates only on that host and not on a groEB host, and vice versa, whereas AgroE-transducing phages were found to plate on either a groEA or a groEB host. Finally, phage T5 did not plate on groEA140 (Zweig and Cummings, 1973) or on groEA140(A~Z~~~)at 32”, whereas it plated with an efficiency of one relative to 594 cells on groE A140(AgroE1cZgs7), a lysogen carrying a prophage of one of the groE transducer isolates. (1) Phage Preparations for Incorporation of Radioactive Methionine The phages were grown lytically and concentrated by centrifugation at 7500 rpm for 16 hr in the GS-3 rotor of the Sorvall centrifuge or by precipitation with polyethylene glycol 6000, as described by Yamamoto et al. (1970). After any of these treatments the phage was resuspended in dilution buffer and was sometimes banded in CsCl density gradients. In other experiments, it was used without further purification. In both cases, however, the preparation was extensively dialyzed against dilution buffer. (m) Preparation of Labeled Extracts of Infected Cells Cells were grown at 37” in RM medium to a density of about 3 x 108/ml. They were harvested by low-speed centrif’ugation and resuspended at a concentration of 10s/ml in RM-Mg medium. If the cells were to be irradiated with ultraviolet light, 6 ml of this

ASSEMBLY

345

suspension was placed in a NO-mm-diameter petri dish and irradiated for about 1 min at 90 ergs/sec/mm2. In both cases, 1 ml of cells was infected at 0” at a m.o.i. of 5 phagesl cell. The mixtures were incubated for 20 min at 0”, diluted with 4 ml of warmed RMMg, and incubated with shaking at 37”. About 150 &i of [35S]Met or 170 Z.&i of a 35S-labeledE. coli hydrolysate, prepared as described by Crawford and Gesteland (1973) (gift of J. Shaw), was added 30 min after the beginning of the incubation at 37”. Incorporation was arrested by the addition of 7 ml of RM medium in the form of crushed ice. For pulse and chase experiments the amount of cells was increased severalfold, according to the number of samples needed. The pulse of [35S]Met was from 30 to 33 min after the beginning of the 37” incubation, and was ended by the addition of a 2000-fold excess of nonradioactive methionine. Samples were withdrawn into crushedice RM medium at different times after the end of the pulse of radioactive methionine. A variation of the above protocol was used in the experiment carried out with groEA97. Here the cells were grown at 33” until a concentration of 7 x lO’/ml. At this point a portion was maintained at 33” while another portion was incubated at 43.5”. The rate of growth at 33 and 43.5” is approximately the same for both 594 and groEA97. When the cells reached a concentration of 3 x 108/ml, their morphology was checked in a light microscope set for phase-contrast optics. The culture of groE A97 cells grown at 43.5“ contained no cells of normal length. Most of the cells were two to three times longer than the cells grown at 33”. The rest of the cells in the 43.5” culture were more than three times longer than the cells in the 33” culture. The control cells, strain 594, grown in an identical way, showed normal, equal length, at both 33 and 43.5” temperatures. Infection, phage adsorption, and further incubation were carried out as already described, except that incubation after the dilution with warm RM-Mg medium was carried out at the same temperature at which the cells were grown prior to harvesting, i.e., cells grown at 33” were incubated at 33” after infection, and cells grown at

346

HELIOS

MURIALDO

43.5” were incubated at 43.5”after infection. The pulse with the hydrolysate containing 35S-labeledamino acids was between 34 and 47 min after dilution into warm medium. The following operations were all carried out at 0”. The radioactive cells were pelleted by centrifugation at 9000 rpm for 15 min in the SS34 rotor of a Sorvall centrifuge. The pellets were resuspended in 1 ml of L buffer, which contains 0.25 M NaCl, 5 mM MgS04, 0.1 mM Na,EDTA, 50 pg/ml of Trasylol, 0.1 mJ4 PMSF, 100 pg/ml of DNase, 10pg/ml of RNase, and 50 miV TrisHCl, pH 7.4, centrifuged again, and resuspended in 0.15 ml of L buffer. Brij-58 solution was added to a concentration of 0.7%. The cells were opened by two or more cycles of freezing in liquid N, and thawing at room temperature until only small pieces of ice remained floating. The tubes were then placed in ice until thawing was complete. The cell debris was removed by centrifugation for 4 min at 15,000 g at 4”. The supernatant was collected and the radioactive components were analyzed by sedimentation. (n) High-Speed Sedimentation Analysis of the Intracellular Radioactive Components One-tenth of a milliliter of cell extract was layered on top of a 3.8-m& lo-30% glycerol gradient in G buffer, which had the same composition as L buffer except that it lacked the enzymes. In some experiments a density cushion of 0.2 ml of CsCl (p = 1.59 gaml-l) or a cushion of 0.2 ml of saturated sucrose in G buffer was positioned at the bottom of the centrifuge tube. (These tubes had been coated by incubating with bovine serum albumin (1 mg/ml) at 45” for 1 hr followed by drying at the same temperature.) Centrifugation was at 56,000 rpm at 2” for various periods of time in the SW60 rotor of a Spinco ultracentrifuge. Fractions were collected from the bottom by puncture of the tube. An appropriate number of drops were collected into a 1.5-ml conical, polypropylene centrifuge tube containing 0.1 ml of E. coli proteins (see below). The radioactivity of a 5-~1 aliquot of these collected fractions was determined in a toluene-Tri-

ton-based butyl-PBD-containing (Pande, 1976).

scintillator

(0) Preparation of E. coli Proteins E. coli proteins were prepared as follows: 5 ml of a late lag phase culture (-2 x log cells/ml) of 159 cells in RM medium was mixed with 6 ml of electrophoresis stacking overlay buffer (Laemmli, 1970), 3 ml of 10% SDS, and 0.5 ml of 2-mercaptoethanol, and incubated for 3 min in a boiling-water bath. The mixture was pipetted up and down several times to reduce viscosity. (p) Polyacrylamide Gel Electrophoretic Analysis of the Polypeptide Composition of the Gradient Fractions Two procedures were used to prepare the fractions for electrophoresis. In the first one, the proteins in the fractions were precipitated at 0” with 10% trichloroacetic acid. After centrifugation the precipitates were washed with acetone and dissolved in 50 ~1 of electrophoresis sample buffer (Laemmli, 1970). After 1 min in a boiling-water bath the samples were layered on the gel for electrophoresis. When a CsCl density cushion was used in the glycerol gradients, the SDS present in the “E. coli proteins” on which the drops of the gradient were collected formed a flaky precipitate that was removed by centrifugation, prior to trichloroacetic acid precipitation. The second method consisted of direct dialysis of each fraction against deionized water in a semimicrodialyzer with a capacity for 24 samples. After dialysis a sample of each fraction was mixed with an equal volume of S2 buffer (0.109 M Tris-HCl, pH 6.8, 5.22% SDS, 17.4% glycerol, 8.7% 2-mercaptoethanol), incubated for 1 min in a boiling-water bath, and layered on the slots in the gels. (q) CkT~ctrophoresis

and Radioautog-

The method of Laemmli (1970) was used for electrophoresis with a stacking gel of 5% and a separating gel of 12.5%. After completion of electrophoresis, the gels were washed with two changes of 500 ml of HZ0

LAMBDA PROHEAD ASSEMBLY

347

for 20 min each. Fixation of the gels with an acetic acid-ethanol mixture was not done because it was found that the intensity of the bands was not different with or without fixation. The gels were dried under vacuum and exposed to Kodak SB-5 X-ray film for l-20 days. Scanning was performed in a Gilford spectrophotometer provided with a linear transport. Care was taken to ensure that the optical density of the bands was within the linear response of the film. RESULTS

The Presence of Oligomeric Structure in hInfected Cells-Sedimentation Studies In order to determine if the assembly of the prohead proceeds via the formation of intermediates smaller than the prohead, I decided to search for phage-specific structures with a sedimentation velocity lower than that of the prohead, but higher than that of the unassembled proteins. This analysis was carried out in wild-type and groE mutant cells, infected with various h mutants. Cells were infected with phages carrying nonsense mutations in each of the prohead genes. In addition to the amber mutations in the morphogenetic genes, the phages carried the b,,, deletion in order to eliminate proteins coded for by the b genetic region, some of which migrate in polyacrylamide gels near several of the morphogenetic gene products of interest (Murialdo and Siminovitch, 1972a). The cells were labeled with [35S]Metlate in phage infection, and at the end of the labeling period, the cells were lysed using a nonionic detergent. After removal of the cellular debris, the extracts were fractionated by sedimentation through glycerol gradients. As may be seen in Fig. 1, high levels of radioactivity were found near the top of such gradients (fractions 19 to 15). These consisted of host material and unassembled proteins of phage origin. Another common feature of the profiles was the presence of a small peak or shoulder at about 25 S (fraction 13). The S value of this peak was determined using as standards E. coli RNA polymerase with an S value of 15 (Burgess, 1971) and A tails with an S value of 45 (Buchwald et al., 1970b;

1

5 fmctlon

10 "umber

15

19

FIG. 1. Sedimentation analysis of labeled extracts of cells infected with A mutants. Cells were phage infected and labeled with a 35S-labeled hydrolysate of E. coli between 29 and 35 min after the start of the 37” incubation. The cell extracts were fractionated in a lo-30% glycerol gradient with a O.&ml cushion of saturated sucrose, by ultracentrifugation at 56,000 rpm for 120 min in a SW60 rotor of a Spinco ultracentrifuge. Nineteen fractions were collected and the radioactivity of a 5-4 sample of each fraction was counted. Sedimentation is from right to left.

Katsura and Kuhl, 1975). The polypeptide components of both these structures are easily observed in the autoradiograms of the SDS gel electrophoretic patterns of the fractions of the glycerol gradients (see, for instance, Fig. 4). Apart from these general features, the profiles obtained from different mutant extracts could be distinguished from each other because extracts from U-- and AC-infected cells exhibited a heterogeneously sedimenting peak of radioactivity near the bottom of the gradient (fractions 1 to 7) which was absent in Eand Nu3- extracts. This heterogeneously sedimenting material presumably represents defective proheads which are known to accumulate in B- and C- extracts but are absent in the E- and Nu.Y extracts (Murialdo and Siminovitch, 1972b; Ray and Murialdo, 1975; Hendrix and Casjens, 1975; Hohn et al., 1975; Becker et al., 1977a). The

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peak of radioactive material sedimenting in the bottom third of the gradient in XE-and ANti--infected cells corresponds to A tails which sediment at 45 S (Buchwald et al., 1970b; Katsura and Ki.ihl, 1975). In order to determine the contribution of phage-induced material to the profiles shown in Fig. 1, the experiment was repeated using cells heavily irradiated with ultraviolet light. This proc’edure markedly suppresses host protein synthesis so that only phage-induced proteins are observed (Murialdo and Siminovitch, 1971). In order to allow the accumulation of prohead intermediates (see Introduction), an amber E mutation plus a second mutation in another prohead gene were introduced into all the phage mutants used in this experiment. As shown in Fig. 2, the 25 S peak (Fig. 1) has now become very prominent because of the

absence of host material. However, when the infecting phage carried a mutation in B or Nu.3 in addition to the Earn mutation, the peak was diminished in height. These results clearly show that at least some of the radioactive material present in the 25 S peak is of phage origin, and that the synthesis of that structure is dependent upon functional genes B and NUS. Identi,fication of A Morphogenetic Proteins in Cell Extracts by Gel Electrophoresis The experiments described in the previous section measuring the distribution of radioactivity in glycerol gradients are only indicative of the presence of multimeric structures. To determine whether these particles contain phage-coded proteins, the extracts of phage-infected cells were fractionated by

a

Fraction

number

FIG. 2. Sedimentation analysis of labeled extracts of uv-irradiated cells infected with A mutants in various prohead genes. The proteins were labeled with [Yl]Met between 30 and 42 min after the beginning of incubation at 37”. The cell extracts were fractionated by sedimentation in a lo-30% glycerol gradient without a saturated sucrose cushion at 56,000 rpm in the SB-405 rotor of a B-60 International ultracentrifuge for 120min. Twenty-four fractions were collected and 5-4 samples counted in a liquid scintillation spectrometer. (a) The profile of radioactivity for cells infected with AEuma,,bo,,cZt, (-) (-) and with ANu-9am.~am81sbne,cIzG(. . .). (b) Cells infected with ACam,,Eam,,,b,,cI,, and with ~Bam,,,Eam,,,b,,cI,, (. . . .). Sedimentation is from right to left.

LAMBDA PROHEAD ASSEMBLY

349

sedimentation, as already described, and 19’75).The bands of the other prohead gene the resulting fractions were analyzed by products present no anomalies. Thus gpNu3 electrophoresis in polyacrylamide gels. and gpE show as single bands of 19K and Ultraviolet-irradiated hosts have been used 38K, respectively. The amber peptides can in previously published studies of A morpho- be visualized in extracts of cells infected with some amber-mutant phages (Fig. 3). genesis in order to reduce the contribution In )&z&am-infected cell extracts, not of hosts bands to SDS-polyacrylamide electrophoretograms. However, since it only does gpNu3 disappear but gpC and the was uncertain whether A prohead assembly gpC-associated bands change mobility to a would proceed normally in such irradiated group of bands called gpC* and gpC*-ascells, it was desirable to identify the prod- sociated bands. This behavior and the MW ucts of A genes B, C, Nu.3, and E in extracts of the different gene C-controlled bands from nonirradiated A-infected cells. In addi- suggest that the gpC- and gpC*-associated tion, it was hoped that use of mutants could bands are generated by successive cleavage provide information about which phage- at only one end of the gpC and gpC* polycoded proteins were essential for complex peptide, respectively. The formation of gpC* from gpC would take place by cleavage at formation. In order to facilitate discussion of the the other end of the polypeptide.’ There is resulting electrophoretograms, we present, a striking difference between the patterns and nonin Fig. 3, a summary of the present knowl- obtained in ultraviolet-irradiated edge of the various bands (proteins) involved irradiated cells with respect to the product in prohead assembly. of gene C. In nonirradiated cells gpC canThe product of gene B usually shows up not be observed in whole extracts, but as as a doublet with an average MW of 61K. will be shown below, some products of gene After infection with a AC amber mutant, C are easily observable in certain fractions several bands disappear from the electro- of glycerol gradients of phage-infected cell phoretograms. The one with the greatest extracts. Similarly, gpC* and gpC*-associMW is referred to as gpC, while the others, ated bands, easily observable in uv-irwhich migrate faster, are called gpC-as- radiated cells (Ray and Murialdo, 1975), are sociated bands. It has been assumed that not seen in whole extracts of nonirradiated the latter are derived from the former by cells, but again some of these gpC*-associated bands can be detected upon fractionaproteolytic cleavage (Ray and Murialdo, tion of the extracts by sedimentation. The rest of the bands, gpB, gpNu3, gpE, and the identifiable amber peptides shown in Fig. 3 show the same behavior whether they are obtained from irradiated or nonirradi-

1% gpNu3 17K gpNqg-

-

-

-

-

-

-

I

FIG. 3. Schematic representation of the bands observed in polyacrylamide gel electrophoresis of the gene products involved in prohead morphogenesis. For an explanation, see text.

1 Recent experiments by J. Shaw and H. Murialdo have shown that gene Nu3 is totally encoded within the rightmost part of gene C (according to the genetic map). Therefore, one nonsense mutation, NuQama8, results in the generation of smaller peptides in both Nu?3 and C gene products. Thus, gpC* does not derive from cleavage of gpC in the absence of gpNu3, as previously suggested (Ray and Murialdo, 1975). Rather gpC* is simply the amber fragment resulting from the presence of the Nu8amag mutation. It should be pointed out that gpC* is active, since AhWam,8 complements ACam,3, which maps close to the N-terminal end of the polypeptide and makes no detectable amber peptide upon cell infection (Jara and Murialdo, 1975; Ray and Murialdo, 1975).

350

HELIOSMURIALDO

ated cells. gpB and some amber peptides of gpB, however, are very difficult to detect in whole extracts of nonirradiated cells (Fig. 4).

The electrophoretograms in Fig. 4 show an analysis of 159 cells infected with the A mutants AB-, kc-, xNu.K, and XE-. The products of genes Nu.3 and E are readily

” 6/

--87K --23K

FIG. 4. Protein components of subassembly complexes. Cells were infected and labeled with a 35S-labeled hydrolysate of E. coli between 29 and 35 min after the start of the 3’7” incubation. The cell extracts were fractionated in a IO-30% glycerol gradient with a 0.2-ml cushion of saturated sucrose by ultracentrifugation at 56,000 rpm for 120 min in a SW60 rotor of a Spinco ultracentrifuge. Nineteen fractions were collected. They were treated as described in Materials and Methods, loaded, and run in a 12.5% acrylamide, SDS-containing gel. Exposure of the dried gels was for 14 days. Sedimentation is fkom left to right. The autoradiograms correspond to cells infected with (a) ABuwz,,&cZ,; (b) hCam,Ad,,; Cc))3\TUSan,b,d~,; (d) ~unzmbnd 26.The fraction number is indicated on top of the autoradiograms; “e” stands for whole extract, prior to fractionation.

LAMBDA

PROHEAD

ASSEMBLY

351

doublet corresponds to gpB since it is absent in B- extracts (track 1, Fig. 4a). The 58K band is probably a host component because it is absent in similar experiments in which the host was irradiated with ultraviolet light prior to phage infection (results not shown). This band will be referred to as h2. The 54.1K and 51.3K bands (which may correspond to gpc” and gpC”‘, respectively; Fig. 3) are derivatives of gpC since they are absent in C- extracts (track 1, Fig. 4b). These last two bands are replaced by bands of 48.5K and 45.7K (which may correspond to gpC*” and gpC*“’ in Fig. 3) in Nz& extracts (track 1, Fig. 4~; bands labeled gpC*). In certain cases, gpNu3 also sediments toward the bottom of the gradient (Figs. 4a and b). This is not surprising for AC--infected cells, since it has been shown that the aberrant proheads that accumulate in this case contain gpNu3 and have an S value between 140 and 160 (Hendrix and Casjens, 1975; Hohn et al., 1975; Ray and Murialdo, 1975). It has been found, however, that the proheads and “monsters” produced in hB-The Composition of the Fast-Sedimenting infected cells do not contain gpNu3 (HenMaterial drix and Casjens, 1975; Hohn et al., 1975). As can be seen from Figs. 4a and b, most The results in Fig. 4a, therefore, suggest of the fast-sedimenting material consists of that gpNu3 sediments rapidly in this case gpE. However, although the concentration not due to an association with gpE but of gpE in cells infected with NG&, AR-, rather by self-association or by association and AC- mutants is comparable (Ray and with another material. It is not clear why Murialdo, 1975), most of the gene E product other proteins, such as gpH and gpH*, remains at the top of the gradient in N?uxwhich are tail proteins are also found at the infected cells. This result indicates that the bottom of the gradient; gpH*, a normal polymerization of gpE is severely impaired component of h tails, also is found in the 45 S or inhibited in the absence of gpNu3 (the fractions (fractions 8-7), coincidental with core protein). other tail proteins such as gpV. It is not However, gpE is not the only phage-coded known if these various fast-sedimenting protein to be found in the fast-sedimenting components are associated with each other, fraction. For example, examination of Fig. or if they are associated with small particu4d shows that bands of 87K, 78K, a doublet late membranous material that could have of average molecular weight 61K, and bands remained in the supernatant fraction of the of 58K, 54. lK, 51.3K, and a few of MW below low-speed centrifugation of the cell extract. 33K also sedimented to the cushion of sucrose at the bottom of the gradient (track Analysis of the 25 S and 30 S Complexes: 1 in Fig. 4d). The two bands of high molecular Effect of Mutations in Genes B, C, weight, 87K and 78K, have not been idenand Nu.3 tified here, but by comparison with previous published observations (Murialdo and As indicated earlier, the absence of the Siminovitch, 1972a; Hendrix and Casjens, NIL?, B, and C gene products affects the 1974b), they probably correspond to gpH nature of the 25 S and 30 S complexes. In and gpH* which are tail proteins. The 61K order to provide a framework for the identifiable in the unfractionated cell extracts (tracks “e” of Figs. 4c and d, respectively). However, gpB is difficult, and gpC essentially impossible, to see (tracks “e” of Figs. 4a and b, respectively). During fractionation by sedimentation, however (Figs. 4 and 5), certain fractions are enriched for particular gene products, making identification of gpB and gpC in the electrophoretograms of the gradient fractions unambiguous. (For instance, see tracks 13 to 11 in Fig. 4d.) Products of the genes involved in prohead assembly were found in three different regions of the gradients: (a) at the top, 2 S-15 S, (b) in the region corresponding to 25 S to 30 S, and (c) at the bottom third of the gradient, %O S. The material at the top of the gradients corresponds to proteins composed of one polypeptide chain or small oligomers and will not be discussed further. The nature of each of the other two regions of the gradient will be examined separately below.

352

HELIOS

analysis of the nature of these complexes under “defective” conditions, we first examined them in detail in E- extracts. In E- extracts the following bands can be observed in the 25 S-30 S region: a doublet of 65K average MW, a second doublet of 61K average MW, and bands of 58K, 54.1K, and 51.3K MW. The 65K doublet is a host component, the product of gene groE (see below). The 61K doublet can be identified as gpB since it is absent both from the 25 S-30 S region and from the fast-sedimenting material in extracts of A.&-infected cells (Fig. 4a). The 58K band corresponds to h2, as discussed above, and the 54.1K and 51.3K bands are under gene C control because they are absent from both the 25 S-30 S region and the fastsedimenting material isolated after infection with a AC- phage (Fig. 4b). Examination of Fig. 4d indicates that the maximum amount of gpgroE is found in fraction 13 (25 S), whereas the maximum of the gpC-associated bands is found in fraction 12 (30 S). For this reason, and for others which will become apparent below, we will consider the 25 S and 30 S complexes as two qualitatively different complexes, the former composed mainly of gpgroE and gpB and the latter composed mainly of gpB and the gpC-associated bands. The material remaining in the 61K position of fraction 13 in Fig. 4a (hB- infection) is of host origin since it was not present in preirradiated cells infected with hB-. It will be referred to as hl. As expected, the gpB doublet was absent from the 25 S and 30 S complexes in Bextracts. However, the sedimentation properties of the gpC-associated bands were also altered under those conditions, since in B- extracts no such bands were found in the 30 S complex. The gpCassociated bands were still synthesized at levels comparable to those found in the h E - infection, however (see fast-sedimenting material in fraction 1 of Figs. 4a and d). In AC--infected cells, not only did the gpC-associated bands disappear from the 30 S component but also gpB failed to sediment as 25 S and 30 S material. However, gpB still sedimented with the fast-sedimenting material (Fig. 4b).

MURIALDO

Thus a functional C-gene product is necessary for association of gpB with both the 25 S and the 30 S complexes and a functional B-gene product for the presence of the gpCassociated bands in the 30 S complex. As may be seen from fraction 13 of Fig. 4a (see Fig. 3 and also Ray and Murialdo (1975)), a 28K doublet is observed in the 25 S complex in B- extracts, presumably due to the presence of the B amber peptide. The fact that the B amber peptide still associates with the 25 S complex suggests that gpB sediments at 25 S due to its association with gpgroE. On the other hand, the fact that mutations in either gene B or gene C abolish the 30 S complex completely might suggest that this latter complex is composed solely of gpB and gpC. Although gpNu3 was never found associated with the 25 S and 30 S complexes, it strongly influenced their composition. In fact, in ANuX-infected cells, gpB disappeared from the 25 s and 30 S components (Fig. 4~). The material remaining in that position corresponds to hl. In addition, gpB was also absent from the fast-sedimenting material, although it is still synthesized at normal rates (compare tracks “e” of Figs. 4c and d; see also Ray and Murialdo (1975)). As can be observed in Fig. 4c, the behavior of the products of gene C was drastically altered in the XNUS- infection. Not only were the gpC-associated bands replaced by the gpC*-associated bands (Fig. 3, Ray and Murialdo, 1975), but all the gene C-controlled peptides disappeared from the 30 S complex (Fig. 4c, fraction 12). As in the case of the Al!- infection, the products of gene C are still synthesized in normal amounts but were found with the fast-sedimenting material (Fig. 4c, fraction 1). These results suggest that an intact NUS gene is essential for the formation of both the 25 S complex containing gpB and the 30 S complex containing gpB and the gpCassociated bands. The results described in this section can be summarized as follows. Two complexes of 25 S and 30 S can be detected in AE-infected cells. The 25 S material is composed mainly of the host component gpgroE and gpB. The 30 S complex is composed of

LAMBDAPROHEADASSEMBLY gpB and the gpC-associated bands. The presence of intact genes B, C, and Nu3 in the infecting phage is essential for the integrity of the 25 S and 30 S material as observed in E- extracts. The Composition of 25 S and 30 S Complexes in Double Amber X-MutantInfected Cells We have presented evidence (Murialdo and Becker, 1978a) that E - extracts are an active source of gpB, gpC, and gpNu3 for synthesis of proheads in vitro. It was of interest, therefore, to compare the compositions of the 25 S and 30 S complexes formed after infection with phage carrying an E - mutation in addition to the mutation of interest. The protocol for this experiment using B-E -, C-E -, and N&‘-E- phages was identical to the one already described using single amber mutants. The data for the E- control were identical to those in Fig. 4d and are not shown here. As expected, the 25 S material in E-B- extracts was composed only of gpgroE and small amounts of hl coincident with the peak of gpgroE (Fig. 5a). No gpC-associated bands were found in the 30 S region of the gradient, although moderate amounts of these products were found in the fast-sedimenting material. These results are similar to those found in the AB- situation (Fig. 4a). It is of interest that the amber fragment generated by the Earn, mutation (33K MW; Hendrix, 1971) also sedimented to the bottom of the gradient, suggesting that this portion of the polypeptide is still able to selfaggregate or to bind to other material. In order to determine if gpNu3 was essential for the cosedimentation of the B amber fragments with gpgroE, extracts of cells infected with AB-Nu3- were analyzed. These results are shown in Fig. 5d. It is clear that while an intact gpNu3 is essential for the sedimentation of gpB to the 25 S position of the gradient, it is totally dispensable for the sedimentation of the Barn,,, fragment(s) to the same position (an interpretation of this observation will be presented in the Discussion). The results of the AE-Nu3- infection (Fig. 5~) also did not differ from those found

353

after ANuS- infection (Fig. 4~). However, there was a significant difference in the behavior of EC- and C- extracts. Whereas gpB did not sediment to the 25 S and 30 S positions in AC--infected cells (Fig. 4b), in AE -C--infected cells there was an accumulation of gpB in the 25 S complex but not in the 30 S complex (Fig. 5b). Therefore, in the absence of gpE, an intact gene C is ~essential only for the formation of the 30 S complex, and not for the formation of the gpgroE-gpB 25 S complex. Not only was gpB present in the 25 S material form in AE -C--infected cells, but the amount seemed to be greater than in the similar fraction from AE --infected cells (Fig. 5b). To verify this observation, comparisons were made of the amount of gpB cosedimenting with gpgroE at 25 S in extracts of cells infected with AE-, hB%-, AC-E-, and ANu.?% - in turn. The amount of gpB was measured by densitometric scanning of the radiolabeled peptides in the autoradiograms (Fig. 6). The tracings confirm the visual impression from Fig. 5 that little gpB is found in the 25 S fraction in extracts from AB-E -- and ANUS-E --infected cells. They also provide a quantitative estimate of the much larger amount of the gpB in AC-E --infected cells as compared to AE -infected cells. The accumulation of gpB in the 25 S complex in AE C--infected cells is associated with the absence of gpB from the 30 S position, suggesting that gpB in the 25 S complex is a precursor of the gpB in the 30 S material. The Host Element in the 25 S Complex: Identijcation of gpgroE Genetic evidence for the involvement of a host component in head assembly has been presented by several authors (Georgopoulos et al., 1972, 1973; Sternberg, 1973). Mutations in the E. coli locus called groE block the morphogenesis of proheads, and A mutants that can overcome this block are located in genes B and E (Georgopoulos et al., 1973; Hohn et al., 1975; Hendrix and Casjens, 1975; Murialdo and Becker, 1978a). Thus, it is possible that gpgroE associates with gpB, albeit transiently, during capsid assembly. Obviously, it was of interest to

HELIOS MURIALDO

FIG. 5. The composition of the 25 S and 30 S complexes in cells infected with A double mutants. Conditions are identical to those in the legend to Fig. 4 except that ultracentrifugation was for 105min. Sedimentation is from left to right. The autoradiograms correspond to cells infected with (a) A.Bam,Eam4b,,,cZ26; (b) hCam,,,Eam,,,b,,,cZ,,; (cl ~uSam.~am,,,b22,cZ,,; and (d) hBamB,JV?&9amasb,,,cI,,. The fraction number is indicated on top of the autoradiograms; “e” stands for whole extract, prior to fractionation.

see if the 65,000 MW peptide in the 25 S phage by in vitro ligation of an EcoRl encomplex is gpgroE. In order to answer this donuclease digest of E. coli DNA with an question, I constructed a groE-transducing equivalent digest of A gti, a generalized

355

LAMBDA PROHEAD ASSEMBLY

- E---. E-B-

-

E-CgpB

--m. E-Nu3-

gp groE i

4

// gP groE

,

69

Molecular

weight

x

63

59

10-3

FIG. 6. The level of gpB cosedimenting with gpgroE. The amount of radioactive material in the bands corresponding to gpgroE and gpB was estimated relative to each other by scanning (as described in Materials and Methods) the optical density of the autoradiogram of the fraction containing the maximum amounts of gpgroE. For scanning, films exposed for only 5 days were used; shorter exposure was used to avoid saturation of the film. The ordinate corresponds to molecular weight.

transducing vector. Following ligation the DNA was packaged into h phage in vitro and plated on groE mutant cells. The putative hgroE~I~~, isolates were checked for the presence of groE by various genetic tests, and one of them, XgroE,cZ,,,, was selected for further study. I next proceeded to determine if the hgroE-transducing phage coded for the 65,000 MW component of the 25 S complex. For this experiment, a culture of strain 159 was divided into two portions. One portion was irradiated with ultraviolet light, as described in Materials and Methods; the second portion was unirradiated. One fraction of the irradiated cells was infected with Xb&,,,, and another fraction was infected with AgroE IcI,,,. The infected cells were pulsed with [35S]Met between 35 and 42 min after the beginning of the incubation at 37”. Unirradiated cells were similarly infected,

but the pulse of radioactivity was added between 25 and 30 min after the beginning of the 37” incubation. The times of radioisotope addition differed in order to compensate for the delay in phage production and decrease in metabolic rate of irradiated cells (Murialdo and Siminovitch, 1971). After the end of the pulse, the cells were broken open. The soluble components were fractionated by sedimentation and the fractions were analyzed by electrophoresis as described earlier (Materials and Methods). The results are presented in Fig. 7. In gel a, where mm-radiated cells were infected with hb#Z,,, the 65,000 MW component can be seen at the 25 S position in fractions 15, 16, and 17. A small amount of gpB can also be observed at the 25 S position, but since there is no block in assembly, under these conditions most of the gpB is presumably utilized for the construction of proheads. The presence

FIG. 7. Identification of gpgroE. Unirradiated cells were infected with phage and labeled with [Vl]Met between 25 and 35 min after the start of the 37” incubation. Irradiated cells were infected and labeled with the same isotope between 30 and 42 min after the beginning of the 37” incubation. The cell extracts, prepared as usual, were fractionated by ultracentrifugation in a lo-30% glycerol gradient with no cushion at the bottom at 56,066 rpm for 150 min in a SW60 Spinco rotor. Twenty-four or twenty-five fractions were collected, dialyzed, and after proper treatment, run in the gels. Exposure after the gels were dried was for 20 days. Sedimentation is from left to right. The gels correspond to (a) mm-radiated cells infected with hbgZ6,,; (b) irradiated cells infected with AbgZ,,; and (c) irradiated cells infected with AgroE,cZe5,. The fraction number is indicated on top of the autoradiograms; “e” stands for total extract, prior to fractionation.

LAMBDAPROHEADASSEMBLY

357

mented at values higher than 25 S; whereas in wild-type cells, a “streak” of gpB sedimenting faster than 25 S together with gpC” and gpC”’ was observed. The significance of this finding is not clear. In any event, this qualitative difference in the sedimentation pattern of the gpB and gpCassociated bands in response to the groE mutations is a clear indication that gpgroE somehow influences the manner of gpB and gpC assembly. For a second series of experiments, we took advantage of the fact that groEA97 cells are temperature sensitive for growth and show a defect in cell fission and form filamentous structures when grown at 43.5”. The defect in cell fission is due to the groE mutation becausein revertants, the properties of growing at high temperature and supporting A growth are recovered simultaneously. Our experiment was therefore performed at the restrictive temperature, under conditions where filament formation occurred. Strain 594 was used as the control. Both cultures were grown at 33” until they reached a concentration of 7 x 107/ ml. A portion was then transferred to a 43.5” bath, while the remainder was kept at 33”. Interaction between gpB and gpgroE After about three more generations (cell Having identified the nature of the com- concentration of 5 X lO*/ml), no normal plexes presumably involved in prohead cells were observed in the 43.5” groEA97 morphogenesis, I next turned to a more de- culture. The cells were then harvested and tailed analysis of the possible interactions infected as described in Materials and between the relevant polypeptides, and to Methods. After infection with AE -C-, the the search for possible intermediates. As a cells grown at 33” were incubated at 33”, first approach to the search for a putative and those grown at 43.5” were incubated gpB-gpgroE interaction, I asked if a muta- at 43.5”. Proteins were labeled with 35S-lation in groE might alter the pattern of sedi- beled protein hydrolysate and the cell exmentation of gpB. Using sedimentation and tracts were analyzed by electrophoresis afelectrophoresis as before, extracts of wild- ter fractionation by sedimentation. The retype cells were compared with those de- sults are presented in Fig. 9. As in the case rived from groEA140 and groEB764 mutant of groEA140 and groEB764 cells, gpgroE cells, before and after infection with E- sedimented as 25 S material even at the rephage. As seen in Fig. 8, gpgroE sediments strictive temperature. The mutation in at 25 S whether or not it is derived from gpgroE therefore blocks its function at 43.5” wild-type cells or from groEA140 or groEB- but not its ability to be assembled into a 25 S 764 mutants (Fig. 8). Furthermore, the structure. It was next of interest to meassedimentation of gpB at 25 S is also not ure the amount of gpB relative to gpgroE affected by these mutations in groE (Fig. 8, in the 25 S complex. This was done by scangels b, c, and d). Minor differences are ob- ning the gel of the peak fractions (fraction servable, however. In the case of the groE 10) and integrating the area under the mutant cells, no gpB was found which sedi- curves. The ratios gpB/gpgroE are shown in

of gpE in all the fractions of the gradient is typical of h wild-type infected cells. It is probably due to the presence of proheads in intermediate states of gpE polymerization or to the partial degradation of almost finished proheads. Proheads cannot be seen in these gradients since they sediment rapidly to the bottom of the tube. Gels b and c of Fig. 7 show the results after infection of irradiated cells with hbfiZ6* and @vE~cZ~~~, respectively. In gel b, the 65,000 MW host component is totally absent from the 25 S position, although gpB and the gpc-associated bands can still be observed at the 30 S position. In gel c, large amounts of the 65,000 MW component are present at the 25 S position, indicating that the groEtransducing phage carries the gene coding for this polypeptide. The results obtained by infecting unirradiated cells with hgroE ,cZ~~, were virtually identical (gel not shown). The results are in agreement with those of Hendrix and Tsui (1978) and Georgopoulos and Hohn (19’78), who have identified a 65,000 MW polypeptide as gpgroE by the use of nonsense and defective mutants.

358

HELIOS MURIALDO

Table 1. These results show that even at low temperatures, less gpB cosediments with gpgroE in groEA9’7 cells than in 594 cells. This 48% reduction in the amount of gpB sedimenting as 25 S is not due to a decrease in the amount of gpB available, since gpB (as well as the other A-induced proteins) appeared to be made in equivalent amounts in the two types of hosts. At high temperatures, there was a drop in the amount of gpB able to cosediment at 25 S relative to the amount found associated at low temperatures. But, whereas in wildtype cells the decrease was about 55%, in groEA97 cells it was of the order of 68%. The above estimates of the reduction of gpB associated with gpgroE represent minimum values because of contamination by the 61K MW hl band in fraction 10 (Figs. 3 and 6).

10 shows that the appearance of radiolabeled gpB in the complex is not very rapid. In fact, no peak was found at the end of the 3-min pulse. By the fourth minute of chase, the amount of label in the complex reached a maximum, and did not change during longer chase times. If the gpB complex acted as an intermediate in prohead assembly, the amount of radioactivity in the 25 S peak would be expected to decrease. However, the pool of unassembled gpB may be too large to detect such a decrease. A similar failure to chase was also observed for phage tails in this analysis, probably due to a flow of label from small molecular weight components (at the top of the gradient) to supramolecular structures, including phage (at the bottom of the gradient tube). DISCUSSION

Kinetics of gpB Complex Formation

The assembly of proheads requires the As a further approach to the study of the function of four phage genes (B,C,Nz.d, E > role of the 25 S complex in prohead mor- and at least one host gene (groE >. The comphogenesis, I examined the rate of forma- plexity of this pathway suggests that astion and disappearance of gpB in the 25 S sembly may proceed through the formation of intermediates smaller in size than the complex in pulse-and-chase experiments. In order to study the entry of label into prohead. In this paper I have reported exthe gpB-containing complex without inter- periments that attempt to identify one or ference from label in gpgroE, the cells were more such intermediates. The results show first irradiated, then infected with Ab221c126, that gpgroE and gpB can be detected in the form of large structures that sediment and pulsed with radioactive methionine from 30 to 33 min after the beginning of rapidly in glycerol gradients. Under conditions in which prohead prethe 37” incubation. The pulse was ended by the addition of a 2000-fold excess of non- cursors accumulate ( hE - infection), a major radioactive methionine to the medium. fraction of gpB sediments as a 25 S comSamples were withdrawn at different times ponent coincident with the sedimentation during the chase period and analyzed by of the host component gpgroE. Some gpB sedimentation. The distribution of radioac- also sediments slightly faster (30 S) than tivity in the gradient was measured. Figure the 25 S component, together with polypep-

FIG. 8. Formation of 25 S and 30 S complexes in uninfected and groE cells. Cells, 594, groEAl40, or groEB764, were grown in RM medium and left uninfected or infected with AEum,,,bZ2,cZ2B.Labeling with [YS]Met was done between 23 and 32 min after the beginning of the 37” incubation. The cell extracts, prepared as usual, were fractionated by ultracentrifugation in a lo-30% glycerol gradient with no cushion at the bottom, at 55,000 rpm for 150 min in a SW-60 Spinco rotor. Between 24 and 25 fractions were collected, dialyzed, and processed for electrophoresis as described in Materials and Methods. After electrophoresis the gels were dried and the film was exposed for 7 days. The autoradiograms correspond to (a) uninfected 594 cells; (b) infected 594 cells; (c) infected groEAl40 cells; and (d) infected groEB’764 cells. Sedimentation is from left to right. The fraction number is indicated on top of the autoradiograms; “e” stands for total extract, prior to fractionation by sedimentation.

359

366

HELIOS MURIALDO

7864

a

a 1

FIG. 9. Formation of 25 S complex in a defective groE- strain. Four cultures of cells-594 grown at 33”, 594 grown at 43.5”, groEA9’7 grown at 33”, and groEA97 grown at 43.5”-were infected with AC~arn,~,Earn~~~b~~,cZ~~, incubated at the same preinfection temperature, and pulsed with a YS-labeled hydrolysate of E. coli as described in Materials and Methods. Cell extracts were prepared and fractionated by ultracentrifugation in a lo-308 glycerol gradient with no cushion at the bottom, at 56,000 rpm for 150 min in a SW-60 Spine0 rotor. The collected fractions were treated and electrophoresed as described in Materials and Methods. After electrophoresis the gels were dried and the film was exposed for 10 days. The autoradiograms correspond to the following infected cells: (a) 594 incubated at 33”; (b) 594 incubated at 43.5”; (c) groEA97 incubated at 33”; and (d) groEA97 incubated at 43.5”. Sedimentation is from left to right. The fraction number is indicated on top of the autoradiograms; “e” stands for total extract, prior to fractionation by sedimentation.

tides gpc” and gpC”’ derived from gpC. This sedimentation pattern most likely refleets the presence of two qualitatively different complexes rather than an overlapping distribution of various species of proteins in the gradient. For example, under the condition of XE -C- infection and h E infection of groE- cells, the sedimentation of gpB at the 25 S position was unaffected,

whereas no gpB was detectable at the 30 S position (see Table 2). (1) ~~~~ of 25 S ad 30 S complexes as Precursor to Proheads As yet there is no direct experimental evidence showing that the 25 S and 30 S complexes are precursors in prohead assem-

LAMBDA PROHEAD ASSEMBLY

bly. In vivo pulse-and-chase experiments designed to demonstrate the flow of label out of the 25 S and 30 S complexes were not successful. This experiment is not conelusive since a similar failure was found for A tails in a pulse-chase experiment, a known normal intermediate in phage assembly (Weigle, 1966). This negative result may be due to the presence of a very large pool of unbound protomers which continuously feed the 25 S and 30 S peaks. A similar problem has been encountered in other phage systems (Roeder and Sadowski, 1977). Because of these difficulties, evidence that the 25 S complex represents a true intermediate may depend on a demonstration that it enters the reaction of prohead assembly in vitro (Murialdo and Becker, 1977, 1978a). However, several indirect lines of evi-

5

10

Fraction

361 TABLE 1

RELATIVE AMOUNT OF gpB COSEDIMENTING WITH gpgroE IN A TEMPERATURE-SENSITIVE groE-

STRAIN

Cell

Temp.

Ratio .sBkrwoE

594 groEA97 594 groEA97

33 33” 43.5” 43.5”

0.356 0.185 0.161 0.059

dence suggest that the gpB in 25 S and 30 S is indeed an intermediate of prohead assembly. The complex is found not only in AE -infected cells but also in wild-type infected cells, suggesting that they are normal intermediates rather than aberrant products

15

20

25

number

FIG. 10. Rate of 25 S gpB complex formation. Irradiated 159 cells were infected with Ab2Z,cIZB and labeled between 30 and 33 min after the start of the 37” incubation with 13?S]Met.At the end of the pulse period a large excess of nonradioactive Met was added to the culture and samples were withdrawn at 1,4, 10, and 16 min after the end of the pulse. Extracts were prepared and fractionated by sedimentation in a lo-30% glycerol gradient containing 0.2 ml of a 1.57 g/ml solution of CsCl as a cushion. Centrifugation was at 55,000 rpm for 120 min. Twenty-six fractions were collected and the radioactivity of a 5~1 sample of each fraction was counted. Sedimentation is from right to left.

362

HELIOS MURIALDO TABLE 2

say for prohead assembly (Murialdo and SEDIMENTATION OF gpB AND gpC DERIVATIVES AT Becker, 1978a). Although all these latter observations THE 25 S AND 30 S POSITIONS IN VARIOUS MUTANT support the idea that the 25 S and 30 S comEXTRACTS plexes represent intermediates, they are gpB in not conclusive. For example, although the gpc” and gpC Ib majority of the intracellular gpB is in the Gene mutated 25 S 30 S in 30 s 25 S and 30 S components, there is a small amount in monomeric form, sedimenting at B the top of the gradient, and also a conc siderable amount in the fast-sedimenting NU.9 material found in fraction 1 of the glycerol E + + + gradients. It is possible therefore that ? POE either of these forms of gpB is used as preEB ++ cursor for prohead assembly, whereas the EC ENuS 25 S and 30 S forms would be due to an aber7 7 7 WT rant dead-end pathway. a (-) The gene product is absent; (+) amount found in the reference situation, that is in AE-, groE+-infected cells; (+ +) increased amounts in respect to the reference situation; (T) decreased amounts in respect to the reference situation.

(2) Function mation

of gpgroE in Complex For-

Several lines of evidence indicate strongly that gpgroE is involved in the formation of the 25 S and the 30 S complexes in association with gpB. First, A phage mutants which made in the absence of gpE. Furthermore, E - extracts contain in active form all the in- can overcome the block in growth imposed gredients (except gpE) necessary for pro- by mutation in the host groE gene, map head assembly in vitro (Murialdo and in genes B and E of the phage (Georgopoulos et al., 1973). This suggests that gpgroE inBecker, 19’78a). In addition, when the formation of the teracts with one or both of these products. gpB-containing 25 S and 30 S complexes is Second, experiments on prohead assembly blocked, morphogenesis of active proheads in vitro have shown that gpB, but not necis also blocked. Thus, it has been shown here essarily gpE, must interact with gpgroE that the presence of a mutation in the host early during prohead assembly. This is be(groE), which is known to block the pro- cause extracts of groE - cells infected with duction of active proheads, results in the ab- AB-Nu3- (gpC and gpE donor) complement sence of gpB and gpC-associated bands in extracts of AC% --infected groE + cells 30 S and a reduction in the amount of gpB (gpB and gpNu3 donor) to form proheads sedimenting at 25 S. This suggests that the in vitro. In contrast, extracts of groE - cells block is exerted at this stage in prohead infected with AC% - do not complement assembly. It is interesting that the aberrant AB-N&?--infected groE + cell extracts (Muparticles that are produced by A infection rialdo and Becker, 1978a). These results of groE mutant cells contain reduced amounts suggest that gpgroE must interact directly of gpB relative to active, wild-type pro- with gpB to give an extract which is capable heads (Hendrix and Casjens, 19’75; Hohn of complementing the AB-NuS-*groE - extract. et al., 1975). It has also been shown here that intact Third, decreased amounts of gpB are gpNu3 is essential for the formation of the found in the 25 S complex in certain groE gpB-containing 25 S and 30 S complexes. mutant-infected cells, and gpB is absent in This result correlates with the observation the 30 S component in some other groEthat Nu3- extracts are not active donors cells. As was the case with AE-C--infected of gpB in the in vitro complementation as- cells, the sedimentation of gpB at 30 S and

LAMBDAPROHEADASSEMBLY higher was prevented during infection of groE mutant cells with AE -. Thus, all of these results show that the sedimentation pattern of gpB is influenced by an interaction with gpgroE. The fact that gpB and gpgroE cosediment in glycerol gradients also suggests that there is a direct association between them. The finding that the Bum, and Barn,,, amber peptides still sediment as 25 S suggests that the complex is formed by association of gpB with other proteins that form the bulk of the complex. Otherwise, it is difficult to explain how a protein, after losing 60% of its mass, can still sediment at the same rate. These data also suggest that the N-terminal 40% of the gpB polypeptide is involved in the binding. It is, therefore, likely that gpB is associated with gpgroE to form the 25 S structure and that its contribution to the mass of this complex is relatively small. The direct demonstration of gpB-gpgroE association might be accomplished by the use of immunoprecipitation techniques and chemical crosslinking agents.

363

complexes (see Fig. 2b). Since the formation of these aberrant proheads is prevented in AC% --infected cells, gpB can accumulate in the 25 S form (Figs. 2d and llc and d). (4) Effect of Nu.3 Mutation Formation

on Complex

The behavior of AN&--infected cells is difficult to explain. N@- extracts contain relatively small amounts of proheads composed exclusively of gpE (Hohn et al., 1975; Hendrix and Casjens, 1975; Ray and Murialdo, 1975). They presumably arise by selfassembly and are biologically inactive (Becker et al., 1977). gpB is not found at 25 S in Nu&- extracts, suggesting that the presence of intact gpNu3 is a prerequisite for formation of the gpB-containing 25 S complex. The formation of the 30 S complex is also prevented in ANti--infected cells. However, since gpNu3 has never been found associated with these putative intermediates in prohead assembly, these findings are difficult to understand. In fact, we have never seen gpNu3 sedimenting into the glycerol gradient in the absence of gpE, (3) Sedimentation Properties of gpB suggesting that there is an interaction beThe sedimentation properties and the rel- tween these two gene products. ative amounts of gpB found in polymeric It is possible that the amber peptide genstructures vary according to the genetic erated by the Ntiam,, mutation interferes makeup of the phage used for infection and with prohead assembly. Under normal cirof the cells infected. A summary of the sedi- cumstances, gpNu3 could act as a bridge bementation properties of gpB and of the gpC tween gpE and the gpB-containing 25 S derivatives is shown in Table 2. As may be complex, resulting in the ultimate formation seen, there is little or no gpB at the 25 S of proheads. The N&am,, peptide, howposition in AB-, XC-, ANu.T, AE-B-, ever, would be able to bind to protomeric AE -NW- infections. Its incorporation into gpB and block the formation of the 25 S the 25 S complex is also partially blocked in gpB-containing complex. This model would groEA97-infected cells. gpB accumulates also explain why a Nu3- extract does not at the 25 S position in AE -- and especially contain active gpB for prohead assembly in in AE-C--infected cells. The reason why vitro (Murialdo and Becker, 1978a). This gpB is not found in 25 S material in C- ex- model is illustrated in Fig. lle. tracts may be because AC--infected cells The behavior of a B- infection is also difproduce abnormal proheads in amounts sim- ficult to understand (Fig. llb). In ABamilar to the normal proheads produced in infected cells, there is an accumulation of AA--infected cells. These inactive proheads biologically inactive proheads (Becker et contain gpB, gpE, and gpNu3 (Hohn et al., al., 1977) which are composed of pX1, pX2, 1975; Hendrix and Casjens, 1975). There- and gpE (Hendrix and Casjens, 1975; Hohn fore, it could be argued that in AC--infected et al., 1975), showing that gpC-gpE assocells gpB is almost entirely bound to aber- ciation and the fusion and cleavage reactions rant proheads and cannot form the 25 S required to form pX1 and pX2 can take place

364

HELIOS

MURIALDO !3PC

a

E-

gpB

gpgroE

l

-

gpB-gpgroE

G5S) -+

gpB-gpC”-gpC”’

(305)

gpge

b

B-

gpBam

+ gpgroE

-

gpBam-gpgoE

gp~E’<+ gpC

l

(255)

0

B-at:-t

gpE

gpNu3 ?

Dx1

;x2 gPE

C

C-

gpB+ gpgroE

gpB-gpgroE

-

(255)

c-aberrant prohead

“\‘<

wNu3 g9roE d

C-E-

gpB + gpgroE

e

Nu3-

gpB + gpNu3am

gpE -

f Wild type

gpB + gpgroE

-

gpB-gpgroE

-

0 -

gpB - gpNu3am Nu3- aberrant prohead

gpB-gpgroE fusion

gPB gpC” gPCgsT3

(255)

&

cleavage reacy+

gpNu3

-+ WE

(255)

WB gpNu3 9pE

(J

+ mmE

gpB - gpC”-gpC”’

( 30 s )

wi4i;;l~

gpNu3’ g* PX2 gPE

FIG. 11. Pathways of A prohead morphogenesis in cells infected with wild-type phages and phages that are mutant in prohead genes. (a-e) Pathways of assembly after infection with E-, Be, C-, C-E-, and NM- phages, respectively. (f) The wild-type prohead assembly pathway. A hyphen between two gene products indicates a physical association between these products. When more than two gene products are listed in this manner, no implication is made regarding nearest neighbor or contact relationships. A large circle denotes the membrane of a prohead-like structure, which may be normal or aberrant. The term aberrant does not refer to the morphology observed in the electron microscope, but rather to the fact that the structure is unable to package DNA. The large hatched circle within the membrane represents a core made up of gpNu3. The small empty ellipse at the base of the membrane is a collar made up of pX1 and pX2 molecules. A filled ellipse represents the connector made up of gpB and gpB*, or the collar and connector, depending on the case. The list of gene products given below a drawn structure summarizes qualitatively the molecular composition of the given structure.

365

LAMBDA PROHEAD ASSEMBLY

in the absence of the wild-type B polypeptide. In this aberrant assembly, the putative 30 S intermediate cannot be seen, although there is indirect evidence for transient interactions involving capsid-assembly elements. Thus, not only do the amber peptides of B cosediment with gpgroE (25 S), but gpgroE function is required for the assembly of the B- aberrant prohead. This gpgroE requirement is manifest by the absence of gpC cleavage and fusion in groEcells infected with a AB mutant (Hohn et al., 1975). Thus, it is possible that the construction of the B- particle is initiated upon the gpgroE complex with which the B amber peptides are associated and that these amber peptides are subsequently lost from the nascent particle. Therefore, the ability of the amber peptides of B to form a 25 S complex may not allow the observation of a truly B- phenotype. Unfortunately no other Barn mutation mapping to the left of the ones used in this study are available.

that gpB and its processed form, gpB*, are located at the connector of the virion (Ray and Murialdo, unpublished experiments; Tsui and Hendrix, 1978), it seems more likely that, as proposed by Murialdo and Ray (1975), pX1 and pX2 are also located at the head-tail connector rather than distributed, one at each corner of the icosahedral shell (Zachary et al., 1977). ACKNOWLEDGMENTS I would like to thank A. Becker for numerous suggestions, extensive help in the preparation of the manuscript, and help in the preparation of A DNA; S. Benchimol for help in in vitro packaging of the recombinant DNA; M. Gold for providing E. coli DNA; P. Ray for help in the development of concepts and ideas in the initial stages of this work; J. Shaw for help in the isolation of the AgroE-transducing phages and for numerous discussions and suggestions; and P. Sadowski and L. Siminovitch for thorough work during the preparation of the manuscript. This work was supported by the M.R.C. of Canada Grant MA-6117 and the NIH Grant GM 24361-01.

(5) Relationship between the 25 S and 30 S Complexes All the conditions that prevent the formation of the 25 S complex also block the appearance of the 30 S complex (Table 2). However, it is possible to demonstrate assembly of the 25 S complex in the absence of the 30 S complex (XC-E --infected cells or AE - infection of groE- cells). Furthermore, in the case of XC% --infected cells, not only is the 30 S complex absent but the 25 S complex contains increased amounts of gpB. This behavior suggests that gpB in the 25 S complex is a precursor of the gpB in the 30 S complex. Although speculative, the putative precursor-product relationship between 25 S and 30 S complexes has been incorporated into the scheme depicted in Fig. 11 (see Figs. lla and f). In any case, since the presence of both intact genes B and C is required for the formation of the 30 S complex containing gpB and gpC derivatives, it seems highly likely that these two gene products interact directly. Therefore, these results strongly suggest that gpB (or gpB*) and the gpC-derived peptides pX1 and pX2 are in close proximity in the prohead and in the virion. Since it is now known

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HELIOS MURIALDO

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