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 earlylysing 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.bacteriophage | latent period | peptide linker T he programmed formation of nonspecific, lethal membrane lesions, or "holes," is featured in many cytocidal phenotypes, including Bax-mediated apoptosis, virion release in infections of nonenveloped mammalian viruses, and the dissemination of important human toxins from bacteria (1-6). The most genetically tractable hole-formation process is host lysis in double-strand DNA bacteriophage infections (7,8). In the infection cycle of phage λ, lysis is a precisely timed event controlled by the holin, S105, a 105-aa product of the S gene (Fig. 1). S105 accumulates in the membrane throughout the morphogenesis period of the infection cycle. This accumulation has no effect on membrane integrity or the proton-motive force (PMF), as shown by noninvasive assays measuring the flagellar rotation speed (9), until suddenly triggering to form irregular holes of unprecedented size (>300 nm) (10). These holes allow release of the phage endolysin, R, from the cytoplasm, resulting in destruction of the cell wall within seconds (9). The holin triggering time is allele specific (11,12), in that it can be advanced or retarded by missense mutations throughout all three transmembrane domains (TMDs) of S105. Holins can be triggered prematurely by energy poisons; for S10...

Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or "holes." In the phages λ and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the λ-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 μm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.bacteriophage | cryoelectron tomography | Escherichia coli | holin | lambda B acteriophage lysis, the most frequent cytolethal event in the biosphere, is a precisely scheduled process controlled by proteins of the holin family (1). Holins are an extremely diverse class of small phage-encoded membrane proteins (2). The best studied holin is S105, a 105-residue polypeptide with three transmembrane domains (TMDs) encoded by the S gene of phage λ (3). Throughout the period of late gene expression and particle assembly, S105 accumulates in the cytoplasmic membrane of Escherichia coli without any effect on its integrity (4). Suddenly, at a programmed time, S105 triggers to form a lesion, or hole, in the membrane; this allows the λ-endolysin, R, to escape from the cytoplasm and attack the cell wall (2). In phages of Gram-negative hosts, there is a third step to complete the lysis pathway involving a protein or protein complex, the spanin, which connects the cytoplasmic and outer membranes (5, 6). In λ, the spanin complex consists of the cytoplasmic membrane protein, Rz, and the outer membrane lipoprotein, Rz1. This complex is essential for lysis in media containing millimolar concentrations of divalent cations, and thus is thought to act by disrupting the outer membrane, possibly by fusion with the inner membrane (6).Although the S105 holin has been extensively studied using genetic and biochemical approaches (3, 4, 7-9), nothing is known about the membrane holes except that they are nonspecific and large enough to allow escape of fully folded tetrameric R-β-galactosi...

Summary Holins control the length of the infection cycle of tailed phages (the Caudovirales) by oligomerizing to form lethal holes in the cytoplasmic membrane at a time dictated by their primary structure. Nothing is currently known about the physical basis of their oligomerization or the structure of the oligomers formed by any known holin. Here we use electron microscopy and single-particle analysis to characterize structures formed by the bacteriophage λ holin (S105) in vitro. In non-ionic or mild zwitterionic detergents, purified S105, but not the lysis-defective variant S105A52V, forms rings of at least two size classes, the most common having inner and outer diameters of 8.5 and 23 nm respectively, and containing approximately 72 S105 monomers. The height of these rings, 4 nm, closely matches the thickness of the lipid bilayer. The central channel is of unprecedented size for channels formed by integral membrane proteins, consistent with the non-specific nature of holin-mediated membrane permeabilization. S105 present in detergent-solubilized rings and in inverted membrane vesicles showed similar sensitivities to proteolysis and cysteine-specific modification, suggesting that the rings are representative of the lethal holes formed by S105 to terminate the infection cycle and initiate lysis.

Summary At a programmed time in phage infection cycles, canonical holins suddenly trigger to cause lethal damage to the cytoplasmic membrane, resulting in the cessation of respiration and the non-specific release of pre-folded, fully active endolysins to the periplasm. For the paradigm holin S105 of lambda, triggering is correlated with the formation of micron-scale membrane holes, visible as interruptions in the bilayer in cryo-electron microscopic images and tomographic reconstructions. Here we report that the size distribution of the holes is stable for long periods after triggering. Moreover, early triggering caused by an early lysis allele of S105 formed approximately the same number of holes, but the lesions were significantly smaller. In contrast, early triggering prematurely induced by energy poisons resulted in many fewer visible holes, consistent with previous sizing studies. Importantly, the unrelated canonical holins P2 Y and T4 T were found to cause the formation of holes of approximately the same size and number as for lambda. In contrast, no such lesions were visible after triggering of the pinholin S2168. These results generalize the hole formation phenomenon for canonical holins. A model is presented suggesting the unprecedentedly large size of these holes is related to the timing mechanism.

Y is the putative holin gene of the paradigm coliphage P2 and encodes a 93-amino-acid protein. Y is predicted to be an integral membrane protein that adopts an N-out C-in membrane topology with 3 transmembrane domains (TMDs) and a highly charged C-terminal cytoplasmic tail. The same features are observed in the canonical class I lambda holin, the S105 protein of phage lambda, which controls lysis by forming holes in the plasma membrane at a programmed time. S105 has been the subject of intensive genetic, cellular, and biochemical analyses. Although Y is not related to S105 in its primary structure, its characterization might prove useful in discerning the essential traits for holin function. Here, we used physiological and genetic approaches to show that Y exhibits the essential holin functional criteria, namely, allele-specific delayed-onset lethality and sensitivity to the energization of the membrane. Taken together, these results suggest that class I holins share a set of unusual features that are needed for their remarkable ability to program the end of the phage infection cycle with precise timing. However, Y holin function requires the integrity of its short cytoplasmic C-terminal domain, unlike for S105. Finally, instead of encoding a second translational product of Y as an antiholin, as shown for lambda S107, the P2 lysis cassette encodes another predicted membrane protein, LysA, which is shown here to have a Y-specific antiholin character. Holins are small phage-encoded membrane proteins that control the length of the bacteriophage infection cycle by determining the time of host lysis (1, 2). Almost all of the experimental work done on holin function at the physiological or molecular level has been focused on a few holin genes: lambda S, phage 21 S 21 , and T4 t. These genes encode the holins S105, S 21 68, and T, which are, respectively, the prototypes of three distinct holin classes, distinguished by their experimentally determined membrane topologies (Fig. 1A). S105 has class I topology, with three transmembrane domains (TMDs) arranged N-out C-in, whereas S 21 68 has class II topology, with two TMDs arranged N-in C-in. As of the last major review, ϳ90% of the Ͼ100 genes from phages and prophages of eubacteria that have been assigned as putative holins fit into either class I or class II, based on predicted topology using widely accepted algorithms (1). These predicted holins are very diverse, with 12 and 14 unrelated sequence families grouped into classes I and II, respectively. Class III topology, with a single TMD and a large periplasmic domain, is restricted to the sequence homologs of protein T, the holin of T4. These proteins are found only in phages of the large myophage class of T4-like phages and in the large siphophage class of T5-like phages.Despite this diversity, holins share some universal functional features, based mainly on extensive work that has been done with these three holin paradigms. Holin genes are turned on at the beginning of viral morphogenesis, so that the holins accumulat...

Crousillac S, Colonna J, McMains E, Dewey JS, Gleason E. Sphingosine-1-phosphate elicits receptor-dependent calcium signaling in retinal amacrine cells. J Neurophysiol 102: 3295-3309, 2009. First published September 23, 2009 doi:10.1152/jn.00119.2009. Evidence is emerging indicating that sphingosine-1-phosphate (S1P) participates in signaling in the retina. To determine whether S1P might be involved in signaling in the inner retina specifically, we examine the effects of this sphingolipid on cultured retinal amacrine cells. Whole cell voltage-clamp recordings reveal that S1P activates a cation current that is dependent on signaling through G i and phospholipase C. These observations are consistent with the involvement of members of the S1P receptor family of G-proteincoupled receptors in the production of the current. Immunocytochemistry and PCR amplification provide evidence for the expression of S1P1R and S1P3R in amacrine cells. The receptor-mediated channel activity is shown to be highly sensitive to blockade by lanthanides consistent with the behavior of transient receptor potential canonical (TRPC) channels. PCR products amplified from amacrine cells reveal that TRPCs 1 and 3-7 channel subunits have the potential to be expressed. Because TRPC channels provide a Ca 2ϩ entry pathway, we asked whether S1P caused cytosolic Ca 2ϩ elevations in amacrine cells. We show that S1P-dependent Ca 2ϩ elevations do occur in these cells and that they might be mediated by S1P1R and S1P3R. The Ca 2ϩ elevations are partially due to release from internal stores, but the largest contribution is from influx across the plasma membrane. The effect of inhibition of sphingosine kinase suggests that the production of cytosolic S1P underlies the sustained nature of the Ca 2ϩ elevations. Elucidation of the downstream effects of these signals will provide clues to the role of S1P in regulating inner retinal function.

The lambda holin, or S105, is a small cytoplasmic membrane protein that controls the timing of host lysis. Using thiol-specific reagents, we determined that the single cysteine residue within S105 was heterogeneously modified during membrane extraction and subsequent immobilized metal ion chromatography. Here we describe the use of a specific and reversible thiol reagent, 2,2′ dithiodipyridine, to generate purified protein with its cysteine residues in the native thiol state. The 2,2′ dithiodipyridine-protection protocol was also successfully used for another, unrelated holin, S2168, and should be generally useful for the purification of membrane proteins.