title: Holin triggering in real time.
abstract: During λ infections, the holin S105 accumulates harmlessly in the membrane until, at an allele-specific time, suddenly triggering to form irregular holes of unprecedented size (>300 nm), releasing the endolysin from the cytoplasm, resulting in lysis within seconds. Here we used a functional S105-GFP chimera and real-time deconvolution fluorescence microscopy to show that the S105-GFP fusion accumulated in a uniformly distributed fashion, until suddenly, within 1 min, it formed aggregates, or rafts, at the time of lethal triggering. Moreover, the isogenic fusion to a nonlethal S105 mutant remained uniformly distributed, whereas a fusion to an early-lysing mutant showed early triggering and early raft formation. Protein accumulation rates of the WT, early, and nonlethal alleles were identical. Fluorescence recovery after photobleaching (FRAP) revealed that the nonlethal mutant and untriggered WT hybrids were highly mobile in the membrane, whereas the WT raft was essentially immobile. Finally, an antiholin allele, S105(ΔTMD1)-mcherryfp, in the product of which the S105 sequence deleted for the first transmembrane domain was fused to mCherryFP. This hybrid retained full antiholin activity, in that it blocked lethal hole formation by the S105-GFP fusion, accumulated uniformly throughout the host membrane and prevented the S105-GFP protein from forming rafts. These findings suggest that phage lysis occurs when the holin reaches a critical concentration and nucleates to form rafts, analogous to the initiation of purple membrane formation after the induction of bacteriorhodopsin in halobacteria. This model for holin function may be relevant for processes in mammalian cells, including the release of nonenveloped viruses and apoptosis.
authors.affiliation: Department of Biology, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: White
authors.forename: Shinobu
authors.initials: S
authors.lastname: Chiba
authors.forename: Ting
authors.initials: T
authors.lastname: Pang
authors.forename: Jill S
authors.initials: JS
authors.lastname: Dewey
authors.forename: Christos G
authors.initials: CG
authors.lastname: Savva
authors.forename: Andreas
authors.initials: A
authors.lastname: Holzenburg
authors.forename: Kit
authors.initials: K
authors.lastname: Pogliano
authors.forename: Ry
authors.initials: R
authors.lastname: Young
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D017209:Apoptosis; D015967:Gene Expression Regulation, Viral; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008027:Light; D021962:Membrane Microdomains; D008856:Microscopy, Fluorescence / Q000379:methods; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D014764:Viral Proteins; C082001:bacteriophage lambda lysis effector protein S105; D049452:Green Fluorescent Proteins