PMID:21187415

Citation	White, R, Chiba, S, Pang, T, Dewey, JS, Savva, CG, Holzenburg, A, Pogliano, K and Young, R (2011) Holin triggering in real time. Proc. Natl. Acad. Sci. U.S.A. 108:798-803
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.
Links	PubMed PMC3021014 Online version:10.1073/pnas.1011921108
Keywords	Alleles; Amino Acid Sequence; Apoptosis; Gene Expression Regulation, Viral; Genes, Dominant; Green Fluorescent Proteins/metabolism; Light; Membrane Microdomains; Microscopy, Fluorescence/methods; Models, Genetic; Molecular Sequence Data; Mutation; Phenotype; Viral Proteins/genetics; Viral Proteins/physiology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
LAMBD:HOLIN		GO:0045918: negative regulation of cytolysis	ECO:0000314:		P		Figure 3. S105, holin, and S107, antiholin, are two transmembrane proteins produced by alternate starts on gene S in phage lambda. S105 accumulates into rafts, which are associated with the formation of large holes in the membrane. These holes allow endolysin to reach the peptidoglycan and result in cytolysis. S105 has three transmembrane domains. The difference between S105 and S107 is an extra N-terminal positive charge on S107, which prevents the first transmembrane domain from entering the membrane. The researchers created a mutant allele of S105 with the first transmembrane domain deleted, such that it would act like S107, and fused it to mCherryFP. Through deconvolution fluorescence imaging, they showed that the fusion protein blocked S105 raft formation, suggesting that antiholins prevent lysis by blocking the formation of holin rafts.	complete CACAO 13115

References

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