Membrane interaction of the portal protein gp20 of bacteriophage T4

Abstract

Assembly of the bacteriophage T4 head structure occurs at the cytoplasmic face of the inner membrane of Escherichia coli with the formation of proheads. The proheads contain an internal scaffolding core that determines the size and the structure of the capsid. In a mutant where the major shell protein gp23 was compromised, core structures without a shell had been detected. Such core structures were also found in the mutant T4am20am23. Since the mutation in gene 20 is at the N terminus of gp20, it was assumed that these core structures assemble in the absence of gp20. However, sequencing showed that the mutation introduces a new ribosome binding site that leads to a restart at codon 15. Although the mutant protein gp20s lacks the very N-terminal sequence, we found that it still binds to the membrane of the host cell and can initiate prohead assembly. This explains its activity to allow the assembly of core structures and proheads at the membrane surface. With a cross-linking approach, we show here that gp20 and gp20s are escorted by the chaperones DnaK, trigger factor, and GroEL and dock on the membrane at the membrane protein YidC.

Fig 1

Sequence analysis of gp20 in T4D and the T4 am20 mutants E208 and E481. The first 16 codons of gp20 of the wild-type T4D (A) and the identical codons of the mutants E208 and E481 (B) are shown. The amber mutation (codon 12) is highlighted in bold, and the ribosome binding sequence (RBS) of gp20s is underlined. The start codon of gp20s is at codon 15. (C) The expression of gp20 and gp20s was analyzed in infected E. coli CR (lanes 1, 3, and 5) and E. coli B (lanes 2, 4, and 6) with an antibody to gp20. To resolve the difference between gp20 and gp20s, a 40-cm-long 7.5% SDS-polyacrylamide gel was used.

Fig 2

Complementation of the T4 am20 mutants by plasmid-borne expression. Diluted phage stocks were spotted onto top agar containing a lawn of E. coli B cells bearing the following plasmids: pT20-40 (A), pT20s-40 (B), pET20-40 (C), and pT20_gfp-40 (D). For a control, E. coli CR and B with no plasmid were tested as plating bacteria (E, F).

Fig 3

Cellular localization of gp20 in uninfected and T4-infected cells. gp20-GFP fusion proteins were expressed in E. coli B for 5 h and examined with fluoromicroscopy (A through C). The plasmid-expressed gp20 (A, C) and the gp20s expressed from E481-infected cells (B) are shown. For a control, GFP alone was expressed under the same conditions (D, E).

Fig 4

Plasmid-encoded gp20 is found in the membrane fraction, whereas gp40 is in the supernatant. E. coli BL21 cultures expressing His-gp20 with gp40 and His-gp20s with gp40 were lysed by a French press and analyzed for soluble and membrane-bound protein (A). The soluble fraction (S) and the membrane fraction (P) were collected by centrifugation and analyzed for their protein content on a Western blot using the respective antibodies. (B) The membrane fraction was placed onto a sucrose step gradient, and fractions 1 to 8 were analyzed for their content of leader peptidase (inner membrane marker protein, top panel), His-gp20 (central panel), and His-gp20s (lower panel) on Western blots.

Fig 5

Membrane binding of gp20. The binding is sensitive to 900 mM NaCl (A). Membrane fractions containing His-gp20 were extracted with the indicated NaCl concentrations and collected by centrifugation. Supernatant (S) and pellet fractions (P) were analyzed. (B) Affinity purification of His-gp20 does not show any comigrating protein. Load fraction (lane 1), flowthrough (lane 2), wash (lane 3), and elution (lanes 4 and 5) are shown.

Fig 6

Cross-linking of His-gp20. E. coli BL21 cells expressing His-gp20 were lysed, and the membrane fraction was treated with 0.6% formaldehyde for 30 min. To resolve the cross-links, the sample was incubated at 95°C for 20 min. To identify the protein bands, mass spectrometry was performed and the peptides were identified. 1, DnaK; 2, YidC; 3, GroEL; 4, Tig; 5, EF-Tu.

Fig 7

Protease mapping of His-gp20 and His-gp20s. E. coli BL21 cells expressing His-gp20 (A) or His-gp20s (B) were converted to spheroplasts and analyzed on a Western blot (lanes 1). To one aliquot, proteinase K (1 mg/ml) was added for 1 h (lane 2), and for another aliquot, the cells were lysed by 3% Triton X-100 and digested by proteinase K for 1 h (lane 3).

Fig 8

Proheads isolated from T4 am20E481-infected cells. (A) E. coli B cells were infected with T4 amE481, and the lysate was analyzed by electron microscopy. Bar = 100 nm. (B) Phage heads (lanes 1 and 5) and proheads from T4 amE481-infected cells (lanes 2 to 4) were purified, and their proteins were analyzed by SDS-PAGE (lanes 1 and 2) and by Western blotting to gp20 (lanes 3 to 5). The gp23 and IPIII proteins were identified by their molecular weight (MW, in thousands) and by mass spectrometry. To resolve the difference between gp20 and gp20s, the Western blot was made from a high-resolution 40-cm SDS-PAGE. Two different amounts of T4 amE481 proheads were applied in lanes 3 and 4.

Fig 9

Assembly intermediates accumulating in T4 am20E481-infected cells. A gallery of proheads showing connected neck structures. Bar = 100 nm.