An assay for vesicle--vesicle fusion involving resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl), the energy donor, and rhodamine, the energy acceptor, has been developed. The two fluorophores are coupled to the free amino group of phosphatidylethanolamine to provide analogues which can be incorporated into a lipid vesicle bilayer. When both fluorescent lipids are in phosphatidylserine vesicles at appropriate surface densities (ratio of fluorescent lipid to total lipid), efficient energy transfer is observed. When such vesicles are fused with a population of pure phosphatidylserine vesicles by the addition of calcium, the two probes mix with the other lipids present to form a new membrane. This mixing reduces the surface density of the energy acceptor resulting in a decreased efficiency of resonance energy transfer which is measured experimentally. These changes in transfer efficiency allow kinetic and quantitative measurements of the fusion process. Using this system, we have studied the ability of phosphatidylcholine, phosphatidylserine, and phosphatidylcholine--phosphatidylserine (1:1) vesicles to fuse with cultured fibroblasts. Under the conditions employed, the majority of the cellular uptake of vesicle lipid could be attributed to the adsorption of intact vesicles to the cell surface regardless of the composition of the vesicle bilayer.

The Lyz endolysin of bacteriophage P1 was found to cause lysis of the host without a holin. Induction of a plasmid-cloned lyz resulted in lysis, and the lytic event could be triggered prematurely by treatments that dissipate the proton-motive force. Instead of requiring a holin, export was mediated by an N-terminal transmembrane domain (TMD) and required host sec function. Exported Lyz of identical SDS͞PAGE mobility was found in both the membrane and periplasmic compartments, indicating that periplasmic Lyz was not generated by the proteolytic cleavage of the membrane-associated form. In gene fusion experiments, the Lyz TMD directed PhoA to both the membrane and periplasmic compartments, whereas the TMD of the integral membrane protein FtsI restricts Lyz to the membrane. Thus, the N-terminal domain of Lyz is both necessary and sufficient not only for export of this endolysin to the membrane but also for its release into the periplasm. The unusual N-terminal domain, rich in residues that are weakly hydrophobic, thus functions as a signal-arrest-release sequence, which first acts as a normal signal-arrest domain to direct the endolysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm. Examination of the protein sequences of related bacteriophage endolysins suggests that the presence of an N-terminal signalarrest-release sequence is not unique to Lyz. These observations are discussed in relation to the role of holins in the control of host lysis by bacteriophage encoding a secretory endolysin.

No abstract

Perhaps the simplest of biological timing systems, bacteriophage holins accumulate during the phage morphogenesis period and then trigger to permeabilize the cytoplasmic membrane with lethal holes; thus, terminating the infection cycle. Canonical holins form very large holes that allow nonspecific release of fully-folded proteins, but a recently discovered class of holins, the pinholins, make much smaller holes, or pinholes, that serve only to depolarize the membrane. Here, we interrogate the structure of the prototype pinholin by negative-stain transmission electron-microscopy, cysteine-accessibility, and chemical cross-linking, as well as by computational approaches. Together, the results suggest that the pinholin forms symmetric heptameric structures with the hydrophilic surface of one transmembrane domain lining the surface of a central channel Ϸ15 Å in diameter. The structural model also suggests a rationale for the prehole state of the pinholin, the persistence of which defines the duration of the viral latent period, and for the sensitivity of the holin timing system to the energized state of the membrane.glycine zipper ͉ holin ͉ lysis ͉ SAR domain ͉ thiol modification

The fate of phage-infected bacteria is determined by the holin, a small membrane protein that triggers to disrupt the membrane at a programmed time, allowing a lysozyme to attack the cell wall. S 21 68, the holin of phage 21, has two transmembrane domains (TMDs) with a predicted N-in, C-in topology. Surprisingly, TMD1 of S 21 68 was found to be dispensable for function, to behave as a SAR (''signal-anchor-release'') domain in exiting the membrane to the periplasm, and to engage in homotypic interactions in the soluble phase. The departure of TMD1 from the bilayer coincides with the lethal triggering of the holin and is accelerated by membrane depolarization. Basic residues added at the N terminus of S 21 68 prevent the escape of TMD1 to the periplasm and block hole formation by TMD2. Lysis thus depends on dynamic topology, in that removal of the inhibitory TMD1 from the bilayer frees TMD2 for programmed formation of lethal membrane lesions.bacteriophage ͉ GxxxG motif ͉ SAR domain ͉ transmembrane domain

Bacteriophage λ has four adjacent genes - S, R, Rz and Rz1 - dedicated to host cell lysis. While S, encoding the holin and antiholin, and R, encoding the endolysin, have been intensively studied, the products of Rz and Rz1 have not been characterized at either the structural or functional levels. Rz1 is an outer membrane lipoprotein and our results indicate that Rz is a type II signal anchor protein. Here we present evidence that an Rz-Rz1 complex that spans the periplasm carries out the final step in the process of host lysis. These results are discussed in terms of a model where endolysin-mediated degradation of the cell wall is a prerequisite for conformational changes in the Rz-Rz1 complex leading to the juxtaposition and fusion of the IM and OM. Fusion of the two membranes removes the last physical barrier to efficient release of progeny virions.

The P1 lysozyme Lyz is secreted to the periplasm of Escherichia coli and accumulates in an inactive membrane-tethered form. Genetic and biochemical experiments show that, when released from the bilayer, Lyz is activated by an intramolecular thiol-disulfide isomerization, which requires a cysteine in its N-terminal SAR (signal-arrest-release) domain. Crystal structures confirm the alternative disulfide linkages in the two forms of Lyz and reveal dramatic conformational differences in the catalytic domain. Thus, the exported P1 endolysin is kept inactive by three levels of control-topological, conformational, and covalent-until its release from the membrane is triggered by the P1 holin.

The phage 21 holin, S 21 , forms small membrane holes that depolarize the membrane and is designated as a pinholin, as opposed to large-hole-forming holins, like S . Pinholins require secreted SAR endolysins, a pairing that may represent an intermediate in the evolution of canonical holin-endolysin systems.For most phages, the termination of each infection cycle is the strictly programmed and regulated lysis of the host, brought about by two phage-encoded proteins (28). One of these, the endolysin, is capable of degrading the cell wall, while the second, the holin, is a small membrane protein which controls endolysin function. During the assembly of progeny virions, holin molecules accumulate in the cytoplasmic membrane without damaging the host. Then, at a time dictated by their primary structure, holins trigger to disrupt the cytoplasmic membrane. For many phages, like and T4, this event releases to the periplasm an endolysin that has accumulated fully folded and enzymatically active in the cytosol. By contrast, phages P1 and 21 encode endolysins that are exported by the host sec system and accumulate in the periplasm as enzymatically inactive proteins tethered to the membrane by an Nterminal SAR (signal anchor-release) domain (25, 26). These SAR endolysins become enzymatically active when their SAR domains exit the membrane to generate the mature, soluble form in the periplasm. This process occurs spontaneously at a low rate but is greatly accelerated when the cytoplasmic membrane is deenergized. Thus, for phages encoding SAR endolysins, holins need only to depolarize the membrane in order to fulfill their role in controlling the timing of lysis. The formation of large membrane lesions like those resulting from S triggering (22) were small and showed a considerable size variation; this heterogeneity persisted when phage from large and small plaques were replated (Fig. 1B to D). Thus, like phage P1 but unlike and T4, the S 21 holin gene is nonessential for plaque formation (7,8,10,27 (Fig. 2A). S 21 and S are not functionally equivalent. We next designed experiments to determine if S 21 68 and R 21 could complement the lysis defect of phages S am R ϩ and S ϩ R am , respectively. Previously, we had reported that, when expressed from the pUC18 derivative pTZ18R, the S 21 gene appeared to be the functional equivalent of S (2). However, the lysis of the culture was not complete even an hour after its onset, despite the fact that the S 21 protein was produced at supraphysiological concentrations from the very-high-copy-number plasmid. For this reason, we repeated these experiments with various alleles of S 21 68 and R 21 transactivated from the late promoter on a medium-copy-number plasmid, in trans to lysisdefective prophages. This system was shown in other studies to support lysis with approximately normal timing (1, 6). As can be seen in Fig. 2B, expression of R 21 from the plasmid complemented the lysis defect of an induced S ϩ R am lysogen, with lysis beginning 55 min after induction and completed within 10 m...