The success of Mycobacterium species as pathogens depends on their ability to maintain an infection inside the phagocytic vacuole of the macrophage. Although the bacteria are reported to modulate maturation of their intracellular vacuoles, the nature of such modifications is unknown. In this study, vacuoles formed around Mycobacterium avium failed to acidify below pH 6.3 to 6.5. Immunoelectron microscopy of infected macrophages and immunoblotting of isolated phagosomes showed that Mycobacterium vacuoles acquire the lysosomal membrane protein LAMP-1, but not the vesicular proton-adenosine triphosphatase (ATPase) responsible for phagosomal acidification. This suggests either a selective inhibition of fusion with proton-ATPase-containing vesicles or a rapid removal of the complex from Mycobacterium phagosomes.

We review data supporting a model in which activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux. The BH3 domain of tBID is not required for targeting but remains on the mitochondrial surface where it is required to trigger BAK to release cytochrome c. tBID functions not as a pore-forming protein but as a membrane targeted and concentrated death ligand. tBID induces oligomerization of BAK, and both Bid and Bak knockout mice indicate the importance of this event in the release of cytochrome c. In parallel, the full pro-apoptotic member BAX, which is highly homologous to BAK, rapidly forms pores in liposomes that release intravesicular FITC-cytochrome c *20A Ê . A definable pore progressed from *11A Ê consisting of two BAX molecules to a *22A Ê pore comprised of four BAX molecules, which transported cytochrome c. Thus, an activation cascade of pro-apoptotic proteins from BID to BAK or BAX integrates the pathway from surface death receptors to the irreversible efflux of cytochrome c.

Endolysosomes can be damaged by diverse materials. Terminally damaged compartments are degraded by lysophagy, but pathways that repair salvageable organelles are poorly understood. Here we found that the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, known to mediate budding and fission on endolysosomes, also plays an essential role in their repair. ESCRTs were rapidly recruited to acutely injured endolysosomes via a pathway requiring calcium and ESCRT-activating factors that was independent of lysophagy. We used live cell imaging to demonstrate that ESCRTs responded to small perforations in endolysosomal membranes and enabled compartments to recover from limited damage. Silica crystals that disrupted endolysosomes also triggered ESCRT recruitment. ESCRTs thus provide a defense against endolysosomal damage likely to be relevant in physiological and pathological contexts.

Chloroquine-resistant Plasmodium falciparum accumulate significantly less chloroquine than susceptible parasites, and this is thought to be the basis of their resistance. However, the reason for the lower accumulation of chloroquine was unknown. The resistant parasite has now been found to release chloroquine 40 to 50 times more rapidly than the susceptible parasite, although their initial rates of chloroquine accumulation are the same. Verapamil and two other calcium channel blockers, as well as vinblastine and daunomycin, each slowed the release and increased the accumulation of chloroquine by resistant (but not susceptible) Plasmodium falciparum. These results suggest that a higher rate of chloroquine release explains the lower chloroquine accumulation, and thus the resistance observed in resistant Plasmodium falciparum.

The BCL-2 family of proteins is composed of both pro-and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-X L monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion eff lux. Release is maximum at ϷpH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl ؊ selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl ؊ -selective (P K ͞P Cl ‫؍‬ 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K ؉ -selective (P K ͞P Cl ‫؍‬ 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K ؉ selectivity (P K ͞P Cl ‫؍‬ 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.