Lysosomes are acidic organelles essential for degradation, signalling and cell homoeostasis. In addition, they play a key role in cell death. Permeabilization of the lysosomal membrane and release of hydrolytic enzymes to the cytosol accompanies apoptosis signalling in several systems. The regulatory mechanism of lysosomal stability is, however, poorly understood. Lipophilic or amphiphilic compounds with a basic moiety will become protonated and trapped within lysosomes, and such lysosomotropic behaviour is also found in many pharmacological drugs. The natural sphingolipid sphingosine exhibits lysosomotropic detergent ability and is an endogenous candidate for controlling lysosomal membrane permeabilization. The lysosomotropic properties of certain detergents might be of use in lysosome-targeting anticancer drugs and drug delivery system in the future. The present review summarizes the current knowledge on the targeting and permeabilizing properties of lysosomotropic detergents from a cellular and physicochemical perspective.

Type IV secretion systems are multiprotein complexes that translocate macromolecules across the bacterial cell envelope. The type IV secretion system in Brucella species encodes 12 VirB proteins that permit this pathogen to translocate effectors into mammalian cells, where they contribute to its survival inside the host. The "core" complex proteins are conserved in all type IV secretion systems, and they are believed to form the channel for substrate translocation. We have investigated the in vitro interactions between the soluble periplasmic domains of three of these VirB components, VirB8, VirB9, and VirB10, using enzyme-linked immunosorbent assays, circular dichroism, and surface plasmon resonance techniques. The in vitro experiments helped in the quantification of the self-association and binary interactions of VirB8, VirB9, and VirB10. Individually, distinct binding properties were revealed that may explain their biological functions, and collectively, we provide direct evidence of the in vitro formation of the VirB8-VirB9-VirB10 ternary complex. To assess the dynamics of these interactions in a simplified in vivo model of complex assembly, we applied the bacterial two-hybrid system in studying interactions between the full-length proteins. This approach demonstrated that VirB9 stimulates the self-association of VirB8 but inhibits VirB10-VirB10 and VirB8-VirB10 interaction. Analysis of a dimerization site variant of VirB8 (VirB8(M102R)) suggested that the interactions with VirB9 and VirB10 are independent of its self-association, which stabilizes VirB8 in this model assay. We propose a dynamic model for secretion system assembly in which VirB8 plays a role as an assembly factor that is not closely associated with the functional core complex comprising VirB9 and VirB10.

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Type IV secretion systems are macromolecular assemblies in the cell envelopes of bacteria that function in macromolecular translocation. Structural biology approaches have provided insights into the interaction of core complex components, but information about proteins that undergo transient interactions with membrane components has not been forthcoming. We have pursued an unbiased approach using peptide arrays and phage display to identify interaction partners and interaction domains of type IV secretion system assembly factor VirB8. These approaches identified the globular domain from the VirB5 protein to interact with VirB8. This interaction was confirmed in cross-linking, pull-down, and fluorescence resonance energy transfer (FRET)-based interaction assays. In addition, using phage display analysis, we identified different regions of VirB6 as potential interaction partners of VirB8. Using a FRET-based interaction assay, we provide the first direct experimental evidence of the interaction of a VirB6 periplasmic domain with VirB8. These results will allow us to conduct directed structural biological work and structure-function analyses aimed at defining the molecular details and biological significance of these interactions with VirB8 in the future.

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca 2؉ pumps. 2؉ -calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.