Molecular function of the dual-start motif in the lambda S holin.
The lambda S gene represents the prototype of holin genes with a dual-start motif, which leads to the synthesis of two polypeptides, S105 and S107. They differ at their N-terminus by only two amino acids, Met-1 and Lys-2, at the beginning of the longer product. Despite the minor difference, the two proteins have opposing functions in lysis, with protein S107 being an inhibitor and protein S105 being an effector of 'hole formation' in the inner membrane. Here, we have studied the molecular mechanism underlying the 'lysis clock' contributed by the dual-start motif. We have used protein fusions in which the secretory signal sequence of the M13 procoat protein VIII has been abutted to the N-terminal Met residues of S105 and S107 respectively. S-dependent 'hole formation' required removal of the signal sequence in both fusion proteins, as both the VIII-S105 and the VIII-S107 fusion proteins were non-functional when leader peptidase cleavage was inhibited. These results strongly supported the hypothesis that functional assembly of S proteins requires translocation of their N-terminus to the periplasm. Using signal sequence cleavage as a measure of translocation, we observed that the translocation kinetics of the N-terminus of the S107 moiety was reduced about threefold when compared with the N-terminus of the S105 moiety. Moreover, depolarization of the membrane resulted in an immediate cleavage of the signal sequence and 'hole formation' exerted by the S107 moiety of the VIII-S107 fusion protein. A model is presented in which S107 with a reversed topology of its N-terminus interacts with S105 and poisons 'hole formation'. Upon depolarization of the membrane, translocation of the N-terminus of S107 to the periplasm results in the functional assembly of S proteins, i.e. 'hole formation'.
Institute of Microbiology and Genetics, Vienna Biocenter, University of Vienna, Dr Bohrgasse 9, 1030 Vienna, Austria.
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Graschopf
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Bläsi
D000595:Amino Acid Sequence; D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004926:Escherichia coli; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D019897:Periplasm; D010539:Permeability; D011190:Potassium Cyanide / Q000494:pharmacology; D021382:Protein Sorting Signals / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
D036022:Capsid Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C108624:S holin, bacteriophage lambda; D014764:Viral Proteins; C082001:bacteriophage lambda lysis effector protein S105; C082002:bacteriophage lambda lysis inhibitor protein S107; C025668:coat protein, Bacteriophage M13; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D011190:Potassium Cyanide