Trimeric intermediate in the in vivo folding and subunit assembly of ...

Proc. Nati Acad. Sci. USA

Vol. 79, pp. 3403-3407, June 1982 Biochemistry

Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22 (protein folding/structural protein/gel electrophoresis)

DAVID GOLDENBERG* AND JONATHAN KING Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Communicated by Robert L. Balduin, February 10, 1982

peptide chains at 390C (10). However, at 300C the mutant polypeptides mature into native tail spikes which are indistinguishable in stability from the wild-type protein (8). These results suggested the existence of essential intermediates in the folding and subunit assembly pathway that are rendered thermolabile by the temperature-sensitive amino acid substitutions. In this paper, we describe a direct analysis of the in vivo folding and assembly of newly synthesized gene 9 polypeptide chains. A similar approach has been used by Ishihama et al. (17) to analyze the assembly of Escherichia coli RNA polymerase in vivo. We have identified an intermediate in the in vivo folding and assembly pathway which is composed of three polypeptide chains that have not completely folded. This species could continue folding in vitro after cell lysis, and it has been tentatively identified by gel electrophoresis.

ABSTRACT Newly synthesized tail spike polypeptide chains mature from trypsin- and NaDodSO4-sensitive unfolded chains to trypsin- and NaDodSO4-resistant native trimers with a t1,2 of 5 min at 300C. A metastable intermediate in subunit folding and assembly was trapped by chilling and isolated by electrophoresis through nondenaturing gels in the cold. A fraction of the intermediate could be matured into native trimers in vitro by incubating at physiological temperature. Mixing experiments with electrophoretically distinct mutant proteins showed that the precursor that matured in vitro represented three tail spike polypeptide chains already associated with each other but not fully folded. Identification of this intermediate reveals that the processes of polypeptide chain folding and subunit assembly are coupled in this large structural protein.

Although numerous investigators have studied the mechanisms by which unfolded proteins refold in vitro (1-3), there have been few experimental studies on the folding and subunit assembly of newly synthesized polypeptide chains in vivo. For some multidomain proteins, such as ,B-galactosidase (4) and immunoglobulins (5), there is evidence that the incomplete polypeptide chains begin to fold as they are synthesized by the ribosome. During the maturation of membrane-associated and secretory proteins, interactions of the polypeptide chain with the membrane may control the folding of the chain (6, 7). Discrete intermediates have recently been identified in the in vitro refolding of RNase and pancreatic trypsin inhibitor (1, 2). We have been interested in the nature of the intermediates in the folding and subunit assembly pathways for large, structurally complex proteins. In an attempt to dissect such processes in vivo, we have carried out a genetic analysis of the maturation of the bacteriophage P22 tail spike endorhamnosidase (8-10). The tail spike is a trimer composed of the 76,000-dalton polypeptide chain coded for by gene 9 of phage P22 (11-14). Six tail spikes assemble irreversibly, but noncovalently, onto the phage head to form a baseplate structure which serves as the cell attachment apparatus. The tail spikes, both free in solution and on the phage particle, possess an endorhamnosidase activity which cleaves the Salmonella 0 antigen during the adsorption process (15, 16). The native trimer is very stable, requiring temperatures >80°C for heat denaturation (8). It is also resistant to denaturation by NaDodSO4 at room temperature and to cleavage by trypsin (14). There are no known post-translational modifications associated with the maturation of the tail spike. Temperature-sensitive mutations have been mapped to >30 sites in gene 9 (9). Characterization of the inactive mutant polypeptide chains synthesized at the restrictive temperature indicates that the temperature-sensitive amino acid substitutions prevent the correct folding or subunit assembly of the poly-

MATERIALS AND METHODS Bacteria and Bacteriophage. Salmonella typhimurium DB7000 was used as the host for all experiments. This strain is suppressor and is therefore restrictive for amber mutants. All of the bacteriophage used here carried amber mutations in genes 5 (amN114) and 13 (amH101). Gene 5 encodes the phage capsid protein. In its absence, no head-related structures are formed, and the tail spike trimer remains free (11, 13). In the absence of the gene 13 product, infected cells do not lyse and phage protein synthesis continues past the normal lysis time. Late after infection by the 5-113- phage, the tail spike polypeptide chain is one of the major proteins synthesized (18). The 9- amber mutant amN110 produces only a small fragment (13,900 daltons) of the gene 9 polypeptide chain (9). The 9- temperature-sensitive mutant used here (tsH304) produces, at the permissive temperature, a native tail spike with an altered electrophoretic mobility, presumably because of a change in net charge (14). Pulse-Chase Labeling Protocol. Cultures of Salmonella DB7000 were grown and infected with bacteriophage as described (8). Sixty minutes after infection, 14C-labeled amino acids (New England Nuclear, NEC-445E) were added to a final concentration of 2 puCi (1 Ci = 3.7 x 101' becquerels) per ml of culture. After a 2-min labeling period, the label was chased by the addition of 50 1,l of 10% solution of acid-hydrolyzed casein per ml of culture. At various times after the addition ofthe unlabeled amino acids, samples were frozen in liquid N2; these samples later were thawed at room temperature to promote cell lysis. For some experiments, unlysed cells were removed by centrifuging at 13,000 x g (Eppendorf centrifuge) for 5 min at 40C.

The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Abbreviation: p9, polypeptide product of gene 9. * Present address: MRC Lab of Molecular Biology, Hills Road, Cambridge, England CB2 2QH.

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Gel Electrophoresis. Gel electrophoresis in the presence of NaDodSO4 was carried out in the discontinuous buffer system of Laemmli (19, 20). Samples for NaDodSO4 electrophoresis were prepared by mixing 2 vol of sample with 1 vol of NaDodSO4 sample buffer to give a final concentration of 2% NaDodSO4. The samples were not warmed above room temperature. Nondenaturing gel electrophoresis was performed by using the discontinuous buffer system of Davis (21) and Ornstein (22). Electrophoresis was carried out at 40C at a constant current of 20 mA (200-400 V) for about 2 hr. The labeled proteins were visualized by preparing autoradiographs on Kodak SB-5 x-ray film. Band intensities were measured with a Joyce-Loebl scanning microdensitometer.

RESULTS Rate of Formation of the Native Trimer. The native tail spike protein is resistant to denaturation by NaDodSO4 at room temperature. Under these conditions, the quaternary structure of the native trimer remains intact, and the native protein migrates more slowly during NaDodSO4 gel electrophoresis than does the NaDodSO4-polypeptide complex prepared from fully denatured protein (14). A pulse-labeling protocol was utilized to measure the kinetics of formation of the NaDodSO4-resistant trimer in vivo. Late after infection, when the tail spike protein is one of the major proteins synthesized, phage-infected bacteria were pulse-labeled with '4C-labeled amino acids. At various times after labeling, samples were frozen in liquid N2. These samples were then

thawed in the presence of NaDodSO4 to induce lysis and electrophoresed through a NaDodSO4/polyacrylamide slab gel. The labeled proteins were visualized by autoradiography. The results from this experiment are shown in Fig. 1. In samples placed in NaDodSO4 immediately after labeling, all of the labeled polypeptide product of gene 9 (p9) migrated in the NaDodSO4 gel as a NaDodSO4-polypeptide complex. In samples lysed at later times, an increasing fraction (up to 60-70%) of the labeled protein migrated at the position of the NaDodSO4-resistant trimer. The formation of the native trimer had a half-time of about 5 min at 30°C. The sum of label in the two bands remained nearly constant after labeling, consistent with a precursor-product relationship between the NaDodSO4sensitive molecules and the native trimer. The NaDodSO4-sensitive precursor molecules might include various possible intermediates in the folding and subunit assembly pathway, such as monomers in various stages of folding, dimers, and trimers not yet completely folded. Analysis of Folding Intermediates by Nondenaturing Gel Electrophoresis. To better characterize the precursor molecules, we attempted to quench the in vivo folding reaction by lowering the temperature rather than by denaturing all of the precursors with NaDodSO4. Lower temperatures have been utilized in the identification of intermediates in the refolding of several other proteins (23, 24, 25). A pulse-chase labeling experiment was performed as described above except that the bacteria were lysed by freezing and thawing in the absence of NaDodSO4. The lysates were promptly chilled on ice and then electrophoresed through a polyacrylamide gel under nondenaturing conditions at 4°C in

T'ime after chase, min 0

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Native trimer

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FIG. 1. Kinetics of in vivo formation of the NaDodSO4-resistant trimer. A culture of S. typhimurium was infected with P22 phage defective in capsid assembly. Sixty minutes after infection, 14C-labeled amino acids were added to the culture; 2 min after that, unlabeled amino acids were added. At the indicated times after the addition of the unlabeled amino acids, samples were withdrawn, mixed with NaDodSO4 sample buffer, and frozen in liquid N2. The samples were later thawed to promote cell lysis, and the lysates were electrophoresed through a NaDodSO4/ polyacrylamide gel. (a) Autoradiograph exposed for 3 days. (b) The gel lanes were scanned with a microdensitometer and the peaks corresponding to the NaDodSO4-polypeptide complex band and the native trimer band were integrated graphically. o, NaDodSO4-polypeptide complex band; e, native trimer band; o, sum of the intensities of the complex band and the native trimer band.

Biochemistry: Goldenberg and King 9+ 9am 9+ 9am 9+ 9am 9+ 9am 9+ I

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Proc. NatL Acad. Sci. USA 79 (1982)

8 4 Time after chase, min

15

order to resolve possible precursors from the native trimer. An autoradiograph from this experiment is shown in Fig. 2. In order to identify the gel bands associated with the tail spike protein and its precursors, a parallel experiment was carried out with an amber mutant ofgene 9 that produces only a small fragment of the p9 polypeptide chain. The position of the native trimer is indicated by the strong band present in the 9+ lysate prepared 30 min after labeling but absent in the parallel 9- am lysate. The appearance of this band in the native gel followed approximately the same kinetics as did the trimer band in the NaDodSO4 gel in Fig. 1. A well resolved band (labeled "B" in Fig. 2) with mobility less than that of the native trimer was found in the 9+ lysates at early times after labeling but was not found in any ofthe 9- am lysates. Band B appeared and then disappeared as the native trimer accumulated, as would be expected of a kinetic intermediate in the formation of the native protein. There was also a rather smeared band (labeled "A") which migrated faster than the native trimer. This species also appeared to be specific to the 9+ lysate and disappeared as the native trimer was formed. Most of the NaDodSO4-sensitive precursor molecules were not well resolved by electrophoresis in the absence of NaDodSO4. This may have been due to heterogeneity or to conformational changes or aggregation during electrophoresis. A third species (labeled "C") was also observed to disappear with time. This band was not specific to the 9+ lysates. However, the possibility that this species is somehow involved in the maturation of the tail spike cannot be ruled out. The bands marked A and B have the properties expected of intermediates in the formation of the native tail spike protein. Electrophoresis in a second dimension in the presence of NaDodSO4 (not shown) revealed that these bands are composed of 76,000-dalton p9 polypeptide chains and are therefore not due to proteolytic degradation. In Vitro Formation ofthe Native Trimer. Productive folding intermediates trapped at low temperature might be able to resume folding to the native trimer when the temperature was increased. Phage-infected bacteria were pulse-labeled as in the previous experiments. Then, at 3 min after labeling, the cells were lysed by freezing and thawing (in the absence of NaDodSO4). The lysate was chilled on ice and centrifuged to remove unlysed cells. Fractions of this supernatant were incubated at 30'C for increasing periods of time and were then chilled on ice. The samples were then electrophoresed through a native gel at 40C, as in the previous section. An autoradiograph of the gel was prepared and the intensities of band B and the native trimer band

30

FIG. 2. Analysis of pulse-labeled precursor molecules by nondenaturing gel electrophoresis. Bacteria infected with 9' or 9- am phage were pulse-labeled for 2 min with '4C-labeled amino acids, followed by a chase with unlabeled amino acids as in Fig. 1. Atthe indicated times, samples of the culture were lysed by freezing and thawing. The lysates were chilled on ice and then electrophoresed through a polyacrylamide gel under nondenaturing conditions at 40C. An autoradiograph was exposed for 3 weeks. The autoradiograph shown was overexposed to allow clear visualization of the bands for intermediates B, A, and C. As a result, the apparent intensities of the native trimer bands do not accurately reflect the time course of the formation of the native trimer. Quantitation of the intensities of the native trimer bands on a less heavily exposed autoradiograph indicated that the time course of formation of the native trimer in this experiment was similar to that in the experiment shown in Fig. 1.

were quantitated with a microdensitometer (Fig. 3). The level ~the'~nativetrimerincreased during-the in viw*o incubation to a level about 50% greater than that in the unincubated sample. The half-time of this reaction was about 4 min. As the level of the labeled native trimer increased during the incubation, the intensity of band B decreased. This decrease also had a halftime of about 4 min. The sum of the intensities ofthe two bands remained constant throughout the incubation. These results are consistent with band B being the species that was converted to the native trimer when the lysate was warmed to 30TC. However, it is also possible that the native trimer was formed in vitro from other precursors that were not resolved by native gel electrophoresis (band A was not resolved in this experiment). The molecules converted to native trimer in vitro represented 20-30% of the labeled precursor molecules. The convertible molecules may be relatively late intermediates in the folding and subunit assembly pathway, and earlier intermediates may not be stable under the conditions of the experiment. I

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FIG. 3. In vitro formation of the native trimer. Phage-infected bacteria were pulse-labeled with "4C-labeled amino acids as in Fig. 1. After a 3-min chase with unlabeled amino acids, the cells were lysed by freezing and thawing. The lysates were chilled, and unlysed cells were removed by centrifuging at 13,000 x g for 5 min. Samples of the supernatant were incubated for the indicated times at 300C and then were chilled on ice. These samples were then electrophoresed through a polyacrylamide gel under nondenaturing conditions at 40C. An autoradiograph was exposed for 3 weeks, and the intensities of the native trimer band and band A were measured with a microdensitometer. o, Band B; e, native trimer band; o, sum of the intensities of band B and the native trimer band.

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In Vitro Subunit Mixing Experiment. If the maturable molecules trapped at low temperatures include monomers or dimers, then the in vitro reaction described above must include a step (or steps) in which the subunits associate. To test for the involvement of free subunits, a mixing experiment was performed using a temperature sensitive mutant (9- tsH304) which produces, at the permissive temperature, a native trimer with an altered electrophoretic mobility (14). Cultures of bacteria were separately infected with 9+ phage and tsH304 phage at 300C and were pulse-labeled with '4C-labeled amino acids. Immediately after labeling, the cells were lysed and supernatants were prepared as in the previous experiments. Samples of the lysates that had either been kept on ice or had been incubated at 30'C were mixed with NaDodSO4 sample buffer at room temperature. A mixture ofthe two lysates was treated identically. The proteins were then electrophoresed through a NaDodSO4 gel and autoradiographed (Fig. 4). When either the 9+ or the tsH304 lysate supernatant was incubated at 30'C, there was a significant increase in the level of the NaDodSO4-resistant trimer. When a mixture of the two supernatants was incubated, the label at the position of both the 9+ trimer and the tsH304 trimer increased. However, there was no formation of species with intermediate mobilities, as would be expected from the formation of hybrid trimers (14). When a lysate supernatant prepared from bacteria coinfected with the 9+ and tsH304 phage was incubated at 30'C, two hybrid species with electrophoretic mobilities intermediate between those of the homogeneous 9+ and tsH304 trimers also formed in vitro, but only if the lysate was prepared from cells coinfected with the two phages. When trypsin (50 ug/ml) was added during the incubation at 30°C, no increase in the label present in the trimer bands was observed. The native trimer is resistant to trypsin digestion (14). However, the precursor molecules, including those that could be converted to the native trimer, were sensitive to trypsin. The trypsin inhibition of the formation of the native trimer also indicates that the precursors were accessible in solution, even

though there was no subunit mixing during the reaction. The results of this experiment indicate that the subunits that formed the native trimer in vitro had already associated within infected cells before lysis. However, these molecules differed from the native trimer in that they were sensitive to NaDodSO4 and to trypsin digestion, indicating that the chains were not completely folded. We have designated this species the "protrimer." Such a partially folded trimer would be expected to migrate more slowly than the native trimer during nondenaturing gel electrophoresis. Thus, the species in band B (Fig. 2), which has the kinetic properties expected of a precursor to the native trimer in both the in vivo and in vitro reactions, has an electrophoretic mobility consistent with its being the protrimer. DISCUSSION By using a pulse-chase labeling protocol in conjunction with gel electrophoresis, we have been able to follow the kinetics of maturation of the native tail spike protein from newly synthesized polypeptide chains in vivo. The half-time of this reaction was about 5 min at 30°C. This rate was considerably slower than the rates reported for the in vitro refolding of small monomeric proteins (26, 27, 28). However, the rates observed for the in vivo folding of the tail spike protein are comparable to the rates of refolding of other oligomeric proteins. The refolding of oligomers such as lactate dehydrogenase (29) and aspartokinase I homoserine dehydrogenase (30) has been reported to have ratedetermining steps with half-times of several minutes. Also, Ito et aL (31) found that the incorporation of newly synthesized subunits into the E. coli RNA polymerase holoenzyme requires up to 15 min in vivo. Note that the observed half-time for the maturation of the tail spike is relatively long compared to the estimated time needed for polypeptide chain synthesis, about 30 sec at 30°C. These experiments did not reveal any evidence for covalent modifications of the polypeptide chain during its maturation. Mixed Ivsates

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FIG. 4. Test for subunit mixing during the in vitro formation of the native trimer. Bacteria were infected at 300C with 9+ or 9- tsH304 phage as indicated. The bacteria were pulse-labeled with 14C-labeled amino acids and lysed immediately after the addition of unlabeled amino acids. The lysates were chilled on ice and centrifuged as in Fig. 3. Samples of the supernatants were mixed with NaDodSO4 without being warmed above 4°C (lanes 0') or were incubated at 300C for 20 min before being mixed with NaDodSO4 (lanes 20'). The samples were then electrophoresed through a NaDodSO4/polyacrylamide gel, and an autoradiograph of the gel was exposed for 7 weeks. The lanes labeled "Mixed lysates" contained a mixture of equal volumes of the 9+ and tsH304 lysate supernatants. The sample in the third lane [marked (+)] of this set was mixed with trypsin (50 ,ug/ ml; Worthington) before being incubated at 300C. wt, Wild type.

Biochemistry: Goldenberg and King In particular, the only two species detected by NaDodSO4 gel electrophoresis were the 76,000-dalton polypeptide chain and the native trimer. Modifications such as proteolytic cleavage or glycosylation might have led to additional species with altered mobilities. However, we cannot rule out the possibility that the observed rate for the formation of the native trimer may reflect the rate of a modification reaction. Also, it is possible that the folding of the tail spike is noncovalently catalyzed, as is believed to be the case for the bacteriophage T4 tail fibers (32). Among the labeled precursor molecules, there was a fraction that could be trapped at 40C in a form that was convertible to the native trimer in vitro when the sample was warmed to 30TC. This reaction did not involve subunit mixing, indicating that the convertible molecules were already trimeric. This trimeric species, which was sensitive to NaDodSO4 and to trypsin, has been named the protrimer. The discrete band (band B in Fig. 2) with a lower mobility than the native trimer during nondenaturing gel electrophoresis has been tentatively identified as the protrimer. The protrimer had the kinetic properties expected of an intermediate in the in vivo formation ofthe native trimer. In vitro, it appeared to be the only species that could continue to fold after cell lysis. The in vitro formation of the native trimer occurred with a rate comparable to the overall rate of folding in vivo. The simplest interpretation ofthese results is that the protrimer is a late intermediate in the productive folding pathway, U IPT ->N and that earlier intermediates are not stable in vitro. Formation of IpT and the subsequent IpT -) N reaction do not differ sharply in rate. Therefore, the formation of N at the beginning of the reaction should be sigmoidal. The data in Fig. 1 are consistent with this. However, it is possible that there are multiple folding pathways and that the protrimer is an intermediate in the folding of only a fraction of the molecules. Thus far, extensive characterization of the molecules that could not continue to fold after cell lysis has not been possible. The intermediates that precede the protrimer might include monomers folded to different degrees and dimers. The diffuse band (band A) in Fig. 2 may represent monomeric precursors. However, we have not been able to resolve this band consistently and have not attempted to characterize it further. Polypeptide chains prepared by dissociating the native trimer tend to aggregate in the absence of a denaturant such as NaDodSO4 or guanidine hydrochloride (unpublished data). The monomeric precursors may form similar aggregates which would prevent them from being resolved by gel electrophoresis and from continuing to fold in vitro. Experiments with the temperature-sensitive mutations in gene 9 indicate that they act at a stage in the folding and subunit assembly pathway prior to the formation of the protrimer (unpublished data). The mutations may block the folding of monomers or the assembly of the monomers into the protrimer. The identification of the protrimer intermediate indicates that the polypeptide chains do not completely fold before assembling into trimers. Rather, the folding of the chains into a

trypsin-resistant form takes place after the chains have associated. Fukuda and Ishihama (33) identified a similar intermediate in the reconstitution of urea-denatured E. coli RNA polymerase. This species was composed of the four subunits of the RNA polymerase core enzyme but was inactive unless warmed from 4°C to 30°C. Kinetic studies of the refolding of other oligomeric enzymes have suggested that the formation of fully active protein depends on intramolecular rearrangements after the subunits associate (3, 30). The maturation of the tail spike trimer also resembles aspects of collagen maturation, in which the triple helical conformation is achieved after initial associa-

Proc. Natl. Acad. Sci. USA 79 (1982)

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tion of procollagen chains through their registration sequences

(35). During the conversion of the protrimer to the native trimer, the protein acquires its resistance to denaturation and to proteolytic digestion. Creighton (2, 34) has suggested that a slow step late in the folding pathway may stabilize the native structure by contributing to a large activation energy separating the folded structure from all unfolded and partially folded conformations. In the case of the tail spike protein, this step appears to occur after the subunits have associated, so that the quaternary structure, as well as the tertiary structure, of the protein is kinetically stabilized during the final stage of folding. We thank Donna Smith for valuable discussions, information, and providing phage strains. This research was supported by Grant PCM7715017 from the National Science Foundation and Grant GM17,980 from the National Institutes of Health. 1. Baldwin, R. L. (1975) Annu. Rev. Biochem. 44, 453-465. 2. Creighton, T. E. (1978) Prog. Biophys. Mol Biol. 22, 231-298. 3. Jaenicke, R. (1979) in Symposium of the 12th FEBS Meeting, Dresden (Pergamon, Oxford), pp. 187-198. 4. Hamlin, J. & Zabin, H. (1972) Proc. Natl. Acad. Sci. USA 69, 412-416. 5. Bergman, L. W. & Kuel, W. M. (1979) J. Biol. Chem. 254, 8869-8876. 6. Wickner, W. (1979) Annu. Rev. Biochem. 48, 23-45. 7. Engelman, D. M. & Steitz, T. A. (1981) Cell 23, 411-422. 8. Goldenberg, D. P. & King, J. (1981)J. Mol Biol. 145, 633-651. 9. Smith, D. H., Berget, P. B. & King, J. (1980) Genetics 96, 331-352. 10. Smith, D. H. & King, J. (1981)J. Mol. Biol 145, 653-676. 11. Israel, J. V., Anderson, T. F. & Levine, M. (1967) Proc. Natl. Acad. Sci. USA 57, 284-291. 12. Botstein, D., Waddel, C. H. & King, J. (1973) J. Mol Biol. 80, 669-695. 13. Berget, P. B. & Poteete, A. R. (1980) J. Virol. 34, 234-243. 14. Goldenberg, D. P., Berget, P. B. & King, J. (1982) J. Biol Chem., in press. 15. Iwashita, S. & Kanegasaki, S. (1973) Biochem. Biophys. Res. Commun. 55, 403-409. 16. Wright, A., McConnell, M. & Kanegasaki, S. (1980) in Virus Receptors, eds. Randall, L. & Phillipson, L. (Chapman and Hall, London), pp. 27-57. 17. Ishihama, A., Taketo, M., Saitoh, T. & Fukuda, R. (1976) in RNA Polymerase, eds. Losick, R. & Chamberlain, M. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 485-502. 18. King, J., Hall, C. & Casjens, S. (1978) Cell 15, 551-560. 19. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 20. King, J. & Laemmli, U. K. (1971) J. Mol. Biol 62, 465-477. 21. Davis, B. J. (1964) Ann. NY Acad. Sci. 121, 404-427. 22. Ornstein, L. (1964) Ann. NY Acad. Sci. 121, 321-349. 23. Hawley, S. A. & Macleod, R. M. (1976) J. Mol. Biol. 103, 655-657. 24. Blum, A. D., Smallcombe, S. H. & Baldwin, R. L. (1978)J. Mol Biol. 118, 305-316. 25. Creighton, T. E. (1980)J. Mol Biol. 137, 61-80. 26. Garel, J.-R., Nall, B. T. & Baldwin, R. L. (1976) Proc. Natl. Acad. Sci. USA 73, 1853-1857. 27. Saxena, V. P. & Wetlaufer, D. (1970) Biochemistry 9, 5015-5023. 28. Stellwagen, E. (1979)J. Mol. Biol 135, 217-229. 29. Rudolph, R., Heider, I., Westhof, E. & Jaenicke, R. (1977) Biochemistry 16, 3384-3390. 30. Garel, J. -R. & Dautry-Varsat, A. (1980) Proc. Natl. Acad. Sci. USA 77, 3379-3383. 31. Ito, K., Iwakura, Y. & Ishihama, A. (1975) J. Mol. Biol. 96, 257-271. 32. Wood, W. B. & King, J. (1979) in Comprehensive Virology, eds. Fraenkel-Conrat, H. & Wagner, R. R. (Plenum, New York), Vol. 13, pp. 581-631. 33. Fukuda, R. & Ishihama, A. (1974) J. Moi. Biol. 87, 523-540. 34. Creighton, T. E. (1979)J. MoL Biol. 129, 411-431. 35. Fessler, J. H. & Fessler, L. I. (1978) Annu. Rev. Biochem. 47, 129-162.