SUMMARY The mitochondrial pathway of apoptosis is initiated by mitochondrial outer membrane permeabilization (MOMP). The BCL-2 family effectors BAX and BAK are thought to be absolute required for this process. Here we report that BCL-2 ovarian killer (BOK) is a bona fide yet unconventional effector of MOMP that can trigger apoptosis in the absence of both BAX and BAK. However, unlike the canonical effectors, BOK appears to be constitutively active and unresponsive to antagonistic effects of the antiapoptotic BCL-2 proteins. Rather, BOK is controlled at the level of protein stability by components of the endoplasmic reticulum–associated degradation pathway. BOK is ubiquitylated by the AMFR/gp78 E3 ubiquitin ligase complex and targeted for proteasomal degradation in a VCP/p97-dependent manner, which allows survival of the cell. When proteasome function, VCP, or gp78 activity is compromised, BOK is stabilized to induce MOMP and apoptosis independently of other BCL-2 proteins.

B‐cell lymphoma 2 (BCL‐2) family proteins mediate mitochondrial apoptosis by regulating mitochondrial outer membrane permeabilization (MOMP), which leads to the activation of the downstream caspase cascade to execute apoptosis. The pro‐apoptotic and anti‐apoptotic BCL‐2 proteins function through protein‐protein interactions in soluble and membrane‐associated states. How soluble BCL‐2 proteins interact is well understood. Anti‐apoptotic proteins, such as BCL‐2 and BCL‐xL, and the pro‐apoptotic effectors of MOMP, including BAK and BAX, interact with pro‐apoptotic BCL‐2 homology 3 (BH3)‐only proteins similarly. Whereas anti‐apoptotic BCL‐2 proteins tightly bind all the BH3‐only proteins to block apoptosis initiation, the effector BCL‐2 proteins are potently triggered by specific BH3‐only proteins to undergo conformational changes, membrane association and insertion, oligomerization, and pore formation. The anti‐apoptotic BCL‐2 proteins also inhibit the activated effectors. p53 is a direct BAX activator inhibited by BCL‐xL, defining a prototype non‐canonical modulator of BCL‐2 proteins‐mediated MOMP. How BCL‐2 proteins cooperate in the presence of membranes remains poorly understood, impeding our understanding of MOMP and apoptosis. Here, we highlight the latest structural views of MOMP by BCL‐2 proteins.

In yeast cells, subunit a of the vacuolar proton pump (VATPase) is encoded by two organelle-specific isoforms, VPH1 and STV1. V-ATPases containing Vph1 and Stv1 localize predominantly to the vacuole and the Golgi apparatus/endosomes, respectively. Ratiometric measurements of vacuolar pH confirm that loss of STV1 has little effect on vacuolar pH. Loss of VPH1 results in vacuolar alkalinization that is even more rapid and pronounced than in vma mutants, which lack all V-ATPase activity. Cytosolic pH responses to glucose addition in the vph1⌬ mutant are similar to those in vma mutants. The extended cytosolic acidification in these mutants arises from reduced activity of the plasma membrane proton pump, Pma1p. Pma1p is mislocalized in vma mutants but remains at the plasma membrane in both vph1⌬ and stv1⌬ mutants, suggesting multiple mechanisms for limiting Pma1 activity when organelle acidification is compromised. pH measurements in early prevacuolar compartments via a pHluorin fusion to the Golgi protein Gef1 demonstrate that pH responses of these compartments parallel cytosolic pH changes. Surprisingly, these compartments remain acidic even in the absence of V-ATPase function, possibly as a result of cytosolic acidification. These results emphasize that loss of a single subunit isoform may have effects far beyond the organelle where it resides.Vacuolar proton-translocating ATPases (V-ATPases) 3 acidify multiple organelles, including mammalian lysosomes, plant and fungal vacuoles, the Golgi apparatus, endosomes, and regulated secretory granules. Through their effects on organelle acidification, V-ATPases impact numerous cellular processes including protein sorting, macromolecular degradation, cytosolic pH and ion homeostasis, and nutrient storage and mobilization (1, 2). Consistent with these diverse roles, complete loss of V-ATPase function is lethal in most organisms. Fungi, however, can tolerate a complete loss of V-ATPase function, and Saccharomyces cerevisiae has emerged as a major model system for mechanistic studies of V-ATPases (3). Yeast mutants lacking V-ATPase activity (vma mutants) show a well defined set of Vma Ϫ growth phenotypes, including sensitivity to high extracellular pH, high Ca 2ϩ concentrations, and heavy metals (4).V-ATPases are highly conserved both at the level of individual subunit sequences and at an overall structural level. A complex of peripheral membrane subunits containing the sites of ATP hydrolysis, V 1 , is attached to an integral membrane complex, V o , containing the proton pore. In higher eukaryotes, many of the subunits are present as multiple isoforms, encoded as multiple genes and/or splice variants (5). These subunit isoforms exhibit tissue-specific expression and/or organelle-specific localization, and in some cases, impart different biochemical characteristics on V-ATPases, possibly tuning their activity to the requirements of different locales (2). Subunit a of the V o sector is present as multiple isoforms in many organisms. Humans have four different subunit a genes (...

The effector B cell lymphoma-2 (BCL-2) protein BCL-2 ovarian killer (BOK) induces mitochondrial outer membrane permeabilization (MOMP) to initiate apoptosis upon inhibition of the proteasome. How BOK mediates MOMP is mechanistically unknown. The NMR structure of the BCL-2 core of human BOK reveals a conserved architecture with an atypical hydrophobic groove that undergoes conformational exchange. Remarkably, the BCL-2 core of BOK spontaneously associates with purified mitochondria to release cytochrome c in MOMP assays. Alanine substitution of a unique glycine in helix α1 stabilizes BOK, as shown by thermal shift and urea denaturation analyses, and significantly inhibits MOMP, liposome permeabilization, and cell death. Activated BID does not activate WT BOK or the stabilized alanine mutant to promote cell death. We propose that BOK-mediated membrane permeabilization is governed in part by its unique metastability of the hydrophobic groove and helix α1 and not through activation by BH3 ligands.

Biosensors can be used in applications ranging from identifying disease biomarkers to detecting spatial and temporal distributions of specific molecules in living cells. A major challenge facing biosensor development is how to functionally couple a biological recognition domain to an output module so that the binding event can be transduced to a visible and quantifiable signal [e.g., Förster resonance energy transfer (FRET)]. Most designs achieve coupling by means of a binding protein that changes conformation upon interacting with its target. This approach is limited by the fact that few proteins possess such natural allosteric mechanisms, and for those that do, the conformational change is frequently not extensive enough to produce a large change in distance between FRET donor and acceptor groups. Here, we introduce protein fragment exchange (FREX) to address both problems. FREX employs two components: a folded binding protein and a fragment duplicated from it, the latter of which can be chosen from many possible fragments. The system is rationally tuned so that addition of ligand induces a conformational change in which the fragment exchanges positions with the corresponding segment of the binding protein. Placing fluorescent donor and acceptor groups on the binding protein and fragment reduces the background level of FRET of the unbound sensor, resulting in a ratiometric FRET response that is expected to be strong and reproducible from protein to protein. FREX is demonstrated using fibronectin III, a monobody binding scaffold that has been tailored to recognize multiple targets. Sensors labeled with Alexa FRET pairs exhibit ratiometric FRET changes of up to 8.6-fold and perform equally well in buffer and serum. A genetically encoded variant of this sensor is shown to be functional in cell lysates and in mammalian cell cultures.