The processing site of gp5 has been determined to be betweenresidues Val-390 and His-391, instead of Ser-351 and Ala-352as previously reported [H . Kanamaru, N . C . Gassner, N . Ye, S.Takeda, and F . Arisaka, J . Bacteriol . 181:2739-2744] . Moreover,the maturation of gp5 is abolished by null mutations in otherhub genes, indicating that cleavage requires the interactionsof several baseplate proteins.

 
gp5 is an essential structural component in the hub of T4 baseplates [10] . Expression of a cloned gene 5 resulted in the synthesis of a protein with a mass of about 63 kDa, consistent with the size predicted from the gene sequence [14] but about 20 kDa greater than the size of gp5 present in the hub . This led to the suggestion that a 20-kDa fragment was proteolytically removed from the gp5 precursor during assembly of the baseplate andthat at least one phage protein was required for processing[14] . A recent report [9] contradicting earlier results [14] suggested that the 75-kDa precursor was synthesized from a cloned gene 5 and that it was cleaved spontaneously or by a host enzyme at residue 351 . To resolve this issue, N-terminal and C-terminal sequence analysis was performed on mature gp5 isolated fromT4 tube baseplates, and polypeptides synthesized from severalcloned truncated derivatives of gene 5 were compared with themature gp5.

N-terminal sequence analysis of the gene products detached from tube baseplates and C-terminal sequence analysis of mature gp5. All the media and buffers used in this study were describedpreviously [1, 2, 19] . Phage tails and tube baseplates wereprepared according to published procedures [15, 21] . Purifiedtube baseplates were treated with 6 M urea at 37°C for 15min or sodium dodecyl sulfate [SDS] sample buffer at 70 and90°C for 3 min each and were subjected to SDS-polyacrylamidegel electrophoresis [PAGE] as described by Laemmli [13] . Ureatreatment overcame the problem of gp5 and gp48 comigrating andbeing difficult to resolve [15] . As shown in Fig . 1, when thetube baseplates were treated with 6 M urea, gp5 as well as gp29,gp27, gp26, gp53, gp3, and gp25 was released from the tube baseplatesbut gp48 was not [Fig. 1, lane A] . Because all hub proteinsexcept gp28, the inside wedge proteins gp53 and gp25 [whichare close to the hub], and the tail tip protein gp3 were releasedfrom baseplates, the results showed that the centers of thebaseplates including the hubs were dissociated during urea treatment.Since gp28 was not detected in this experiment, the resultsindicated that gp28 is not a hub structural protein, as reportedpreviously [10, 11] . When the tube baseplates were treated with SDS buffer at 70°C, two bands appeared in the 24-kDa region[Fig. 1, lane B], the upper band being gp11 and the lower band being gp26 [confirmed by N-terminal sequence analysis] [Table 1] . When the tube baseplates were treated at 90°C, the gp26 band comigrated with gp11 [Fig . 1, lane C] . All the observedmolecular weights of the gene products are close to the valuescalculated except for gp5 because of its processing . After electrophoresis,the proteins were transferred from the gel to a polyvinylidenedifluoride [PVDF] membrane [20], and the bands of interest weresubjected to automatic Edman degradation for N-terminal sequenceanalysis . The N-terminal sequences are almost identical to theDNA-deduced sequences except that the five N-terminal aminoacid residues [MYEYK] are removed from gp26 [Table 1] . Met-1is retained or removed, consistent with reference 3 . It wasreported that the eight N-terminal residues of gp11 were cleaved[5] and gp26 was not located in the baseplate [10], but the present results showed that only Met-1 of gp11 is removed and gp26 is a component of the tail . One reason for the discrepancymay be interference from gp26, which comigrates with gp11 [Fig. 1, lane C].

 
For C-terminal sequence analysis of mature gp5, the gels werestained after electrophoresis [22] and gp5 was extracted from the excised bands [43 kDa] with a 50 mM [NH₄]HCO₃ solution containing1% SDS at 4°C for 12 h three times . Approximately 680 pmol[quantitative analysis as described in reference 17] of gp5was extracted from the gels and incubated with 1.2 U of carboxypeptidase[Sigma] at 25°C . The incubation was stopped [7] at 0, 15,30, and 45 min . One cycle of Edman degradation was carried outfor each sample . The results showed that carboxypeptidase digestionproduced Val, Tyr, Pro, Tyr, and Glu in sequence, and all PTHamino acid peaks showed equivalent molar yields . There was onlyone sequence, Glu-Tyr-Pro-Tyr-Val, at positions 386 to 390,consistent with that deduced from the DNA sequence . None ofthe peaks corresponding to amino acids adjacent to the C-terminalSer-351 reported in reference 9 were found . These findings indicatedthat the mature gp5 has a C-terminal Val-390, and the cleavageoccurs between Val-390 and His-391, not between Ser-351 andAla-352 . The calculated molecular weight of 42,900 is consistentwith the observed value, 42,000 to 43,000 . The calculated isoelectricpoint of the mature gp5 was close to 8, similar to the reportedvalue [15].

Expression and detection of gp5 polypeptides. Three truncated derivatives of gene 5 were cloned [16] . After induction, they produced gp5 derivatives with C-terminal Ser-360, Val-380, or Val-390 [18] . Because the pET-17b vector used inthis study expresses an N-terminal fusion protein, the ribosomalbinding site and other unnecessary sequences were removed fromthe vector by restriction with XbaI and EcoRI . The XbaI site and ribosome binding site sequences were included in the 5' primer upstream of the gene 5 start codon . The stop codon andthe EcoRI site were included in three distinct 3' primers forPCR amplification of the three gene 5-truncated derivativesthat expressed 360, 380, or 390 amino acid polypeptides . T4dcDNA [Takara, Kyoto, Japan] was used as a template for PCR.The primers were synthesized by Bio/Can Scientific or AppliedBiosystems Japan Inc . The three gp5-derived polypeptides wereexamined by Western blot analysis [6, 20] by using antiserum against gp5 [maintained and used in this laboratory] . As shown in Fig . 2, the gp5 C-terminal Val-390 derivative comigrated with mature gp5 obtained from tube baseplates, consistent with the assignment of this cleavage site in gp5.

 
Examination of the processed gp5 from amber lysates. To identify the T4 gene products responsible for gp5 processing,T4 amber mutants with mutations in the baseplate genes wereused to infect Escherichia coli BE [sup⁰] for preparation of amber lysates [1, 15], and mature gp5 was detected by Westernblot analysis . We first interpreted the upper band in the blots[Fig . 3 and 4] as the uncleaved gp5 precursor or, perhaps, across-reacting E . coli protein . The results shown in Fig . 4 reveal that this band is gp23 because it has the correct size,49 kDa [4], and it did not appear in the 23^– lane [Fig. 4, lane G] . This indicated that gp23 has some amino acid sequencessimilar to gp5, because gp23 cross-reacted, as shown in Table2 and Fig . 5 . Since only the 26am N131 lysate gave rise to maturegp5, these results suggested that gp5 processing is a resultof the interactions of several baseplate gene products and doesnot involve an E . coli enzyme or autolysis . If the processingoccurred either via an E . coli enzyme or autolytically, themature gp5 should be detected in all lysates [except for gene5 amber lysate]; however, this was not the case . On the otherhand, because the gene 26 amber mutant is unable to form hubs[10] and the mature gp5 was detected in this lysate, gp5 processingmay occur before hub formation . This suggests that gp5 entersthe hub pathway after processing.

 
Western blot analysis of gp26. The assignment of gp26 as a band in SDS-PAGE had some ambiguitybecause gp26 comigrated with gp11 [Fig . 1] . To identify gp26on SDS-PAGE, an antiserum against a gp26 C-terminal peptidewas prepared according to Iwai et al . [8], and Western blotanalysis was carried out . As shown in Fig . 6, the 24-kDa band was found to be gp26, which was confirmed by N-terminal sequence analysis [Table 1] . The molecular mass of gp26 was similar to that deduced from the nucleotide sequence, not 41 kDa as reported [12], and it is unlikely that the peptide cleaved from precursorgp5 was transferred to gp26 [14].

 
In conclusion, these results demonstrate that gp5 is processed proteolytically, with a cleavage between Val-390 and His-391, resulting in the loss of 20 kDa and the conversion of the 63-kDa precursor gp5 to the 43-kDa mature gp5 found in the baseplate,and support the earlier sug-estion [14] that processing of gp5 depends on other T4 genes.

 
* Corresponding author . Present address: Nansho Kano, Department of Ecology, Malcom Co., Ltd., 15-10 Honmachi 4-chome, Shibuya-ku, Tokyo 151-0071, Japan . Phone: 81 3 3320 5611 . Fax: 81 3 3320 5615 . E-mail: nyeco@hotmail.com .

1. Arisaka, F., T . Nakako, H . Takahashi, and S.-I . Ishii. 1988 . Nucleotide sequence of the tail sheath gene of bacteriophage T4 and amino acid sequence of its product . J . Virol . 62:1186-1193.
2. Arisaka, F., S . Takeda, K . Funane, N . Nishijima, and S . Ishii. 1990 . Structural studies of the contractile tail sheath protein of bacteriophage T4 . 2 . Structural analysis of the tail sheath protein, Gp18, by limited proteolysis, immunoblotting, and immunoelectron microscopy . Biochemistry 29:5057-5062.
3. Ben-Bassat, A., and K . Bauer. 1987 . Amino-terminal processing of proteins . Nature 369:315.
4. Black, L . W., M . K . Showe, and A . C . Steven. 1994 . Morphogenesis of the T4 head, p . 218-258 . In J . D . Karam [ed.], Molecular biology of bacteriophage T4 . ASM Press, Washington, D.C.
5. Coombs, D . H., and F . Arisaka. 1994 . T4 tail structure and function, p . 259-281 . In J . D . Karam [ed.], Molecular biology of bacteriophage T4 . ASM Press, Washington, D.C.
6. Duda, R . L., M . Gingery, and F . A . Eiserling. 1986 . Potential length determiner and DNA injection protein is extruded from bacteriophage T4 tail tubes in vitro . Virology 151:296-314.
7. Hayashi, R. 1983 . Carboxypeptidase Y protein . Nucleic Enzyme Exp . Lecture 28:1421-1431 . [In Japanese.]
8. Iwai, K., S.-I . Fukuoka, T . Fushiki, K . Kido, Y . Sengoku, and T . Semba. 1988 . Preparation of a verifiable peptide-protein immunogen: direction-controlled conjugation of a synthetic fragment of the monitor peptide with myoglobin and application for sequence analysis . Anal . Biochem. 171:277-282.
9. Kanamaru, H., N . C . Gassner, N . Ye, S . Takeda, and F . Arisaka. 1999 . The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage . J . Bacteriol . 181:2739-2744 .
10. Kikuchi, Y., and J . King. 1975 . Genetic control of bacteriophage T4 baseplate morphogenesis . II . Mutants unable to form the central part of the baseplate . J . Mol . Biol . 99:673-694.
11. Kikuchi, Y., and J . King. 1975 . Genetic control of bacteriophage T4 baseplate morphogenesis . III . Formation of the central plug and overall assembly pathway . Mol . Gen . Genet . 99:695-716.
12. Kozloff, L . M., and M . Lute. 1984 . Identification of bacteriophage T4D gene products 26 and 51 as baseplate hub structural components . J . Virol . 52:344-349.
13. Laemmli, U . K. 1970 . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227:680-685.
14. Mosig, G., G . W . Lin, J . Franklin, and W.-H . Fan. 1989 . Functional relationships and structural determinants of two bacteriophage T4 lysozymes: a soluble [gene e] and a baseplate-associated [gene 5] protein . New Biol . 1:171-179.
15. Nakagawa, H., F . Arisaka, and S.-I . Ishii. 1985 . Isolation and characterization of the bacteriophage T4 tail-associated lysozyme . J . Virol . 54:460-466.
16. Sambrook, J., E . F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual, 2nd ed . Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
17. Smith, P . K., R . I . Krohn, G . T . Hermanson, A . K . Mallia, F . H . Gartner, M . D . Provenzano, E . K . Fujimoto, N . M . Goeke, B . J . Olson, and D . C . Klenk. 1985 . Measurement of protein using bicinchoninic acid . Anal . Biochem . 150:76-85.
18. Tabor, S., and C . C . Richardson. 1985 . A bacteriophage T7 RNA polymerase / promoter system for controlled exclusive expression of specific genes . Proc . Natl . Acad . Sci . USA 82:1074-1078.
19. Takeda, S., F . Arisaka, S.-I . Ishii, and Y . Kyogoku. 1990 . Structural studies of the contractile tail sheath protein of bacteriophage T4 . 1 . Conformational change of the tail sheath upon contraction as probed by differential chemical modification . Biochem . 29:5050-5056.
20. Towbin, H., T . Staehelin, and J . Gordon. 1979 . Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications . Proc . Natl . Acad . Sci . USA 76:4350-4354.
21. Tshopp, J., F . Arisaka, R . V . Driel, and J . Engel. 1979 . Purification, characterization and reassembly of the bacteriophage T4D tail sheath protein P18 . J . Mol . Biol . 128:247-258.
22. Wyckoff, M., D . Rodbard, and A . Chrambach. 1977 . Polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers using multiphasic buffer systems: properties of the stack, valid R_f-measurement, and optimized procedure . Anal . Biochem . 78:459-482.

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