Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death

It was recently demonstrated that during apoptosis, active caspase 9 and caspase 3 rapidly accumulate in the mitochondrion-enriched membrane fraction (D. Chandra and D. G. Tang, J. Biol. Chem.278:17408-17420, 2003). We now show that active caspase 8 also becomes associated with the membranes in apoptosis caused by multiple stimuli. In MDA-MB231 breast cancer cells treated with etoposide (VP16), active caspase 8 is detected only in the membrane fraction, which contains both mitochondria and endoplasmic reticulum (ER), as revealed by fractionation studies. Immunofluorescence microscopy, however, shows that procaspase 8 and active caspase 8 predominantly colocalize with the mitochondria. Biochemical analysis demonstrates that both procaspase 8 and active caspase 8 are localized mainly on the outer mitochondrial membrane (OMM) as integral proteins. Functional analyses with dominant-negative mutants, small interfering RNAs, peptide inhibitors, and Fas-associated death domain (FADD)- and caspase 8-deficient Jurkat T cells establish that the mitochondrion-localized active caspase 8 results mainly from the FADD-dependent and tumor necrosis factor receptor-associated death domain-dependent mechanisms and that caspase 8 activation plays a causal role in VP16-induced caspase 3 activation and cell death. Finally, we present evidence that the OMM-localized active caspase 8 can activate cytosolic caspase 3 and ER-localized BAP31. Cleavage of BAP31 leads to the generation of ER- localized, proapoptotic BAP20, which may mediate mitochondrion-ER cross talk through a Ca(2+)-dependent mechanism.     

Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals,enhancing cytochrome c release to the cytosol

Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.     

BAP31 is involved in the retention of cytochrome P450 2C2 in the endoplasmic reticulum

Microsomal cytochrome P450 2C2 is an integral endoplasmic reticulum (ER) membrane protein that is directly retained in the ER and excluded from transport vesicles. We have used bimolecular fluorescence complementation and co-immunoprecipitation to show that a ubiquitous ER membrane protein (BAP31) interacts with P450 2C2 in transfected COS-1 cells. A chimera containing only the N-terminal signal anchor of P450 2C1 (P450 2C1-(1-29)) also interacted with BAP31, which is consistent with interaction of the two proteins via their transmembrane domains. Down-regulation of BAP31 expression with small interfering RNA resulted in redistribution of green fluorescent protein-tagged P450 2C2 or P450 2C1-(1-29) from the ER into the nuclear membrane and compact perinuclear compartment structures as well as the cell surface in a small fraction of the cells. In Bap31-null embryonic stem cells, a significant fraction of P450 2C2 or P450 2C1-(1-29) was detected at the cell surface and nuclear envelope, but was redistributed to the ER by expression of BAP31. The expression level of P450 2C2 was significantly increased in COS-1 cells with repressed levels of BAP31. Formation of the pro-apoptotic p20 fragment of BAP31 was detected in transfected COS-1 cells expressing P450 2C2, and annexin V staining was consistent with the activation of an apoptotic pathway in these cells. Down-regulation of BAP31 with small interfering RNA partially reversed the apoptosis. These results suggest that interaction of P450 2C2 with BAP31 is important for its ER retention and expression level and that BAP31 may be involved in the regulation of apoptosis induced by the ER overload response to increased expression of P450.     

Structural and Biophysical Characterization of the Cytoplasmic Domains of Human BAP29 and BAP31

Two members of the B-cell associated 31 (BAP31) family are found in humans; BAP29 and BAP31. These are ubiquitously expressed receptors residing in the endoplasmic reticulum. BAP31 functions in sorting of membrane proteins and in caspase-8 mediated apoptosis, while BAP29 appears to mainly corroborate with BAP31 in sorting. The N-terminal half of these proteins is membrane-bound while the C-terminal half is cytoplasmic. The latter include the so called variant of death effector domain (vDED), which shares weak sequence homology with DED domains. Here we present two structures of BAP31 vDED determined from a single and a twinned crystal, grown at pH 8.0 and pH 4.2, respectively. These structures show that BAP31 vDED forms a dimeric parallel coiled coil with no structural similarity to DED domains. Solution studies support this conclusion and strongly suggest that an additional α-helical domain is present in the C-terminal cytoplasmic region, probably forming a second coiled coil. The thermal stability of BAP31 vDED is quite modest at neutral pH, suggesting that it may assemble in a dynamic fashion in vivo. Surprisingly, BAP29 vDED is partially unfolded at pH 7, while a coiled coil is formed at pH 4.2 in vitro. It is however likely that folding of the domain is triggered by other factors than low pH in vivo. We found no evidence for direct interaction of the cytoplasmic domains of BAP29 and BAP31.     

Caspase-resistant BAP31 inhibits fas-mediated apoptotic membrane fragmentation and release of cytochrome c from mitochondria

BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two identical caspase recognition sites (AAVD.G) that are preferentially cleaved by initiator caspases, including caspase 8. Cleavage of BAP31 during apoptosis generates a p20 fragment that remains integrated in the membrane and, when expressed ectopically, is a potent inducer of cell death. To examine the consequences of maintaining the structural integrity of BAP31 during apoptosis, the caspase recognition aspartate residues were mutated to alanine residues, and Fas-mediated activation of caspase 8 and cell death were examined in human KB epithelial cells stably expressing the caspase-resistant mutant crBAP31. crBAP31 only modestly slowed the time course for activation of caspases, as assayed by the processing of procaspases 8 and 3 and the measurement of total DEVDase activity. As a result, cleavage of the caspase targets poly(ADP-ribosyl) polymerase and endogenous BAP31, as well as the redistribution of phosphatidylserine and fragmentation of DNA, was observed. In contrast, cytoplasmic membrane blebbing and fragmentation and apoptotic redistribution of actin were strongly inhibited, cell morphology was retained near normal, and the irreversible loss of cell growth potential following removal of the Fas stimulus was delayed. Of note, crBAP31-expressing cells also resisted Fas-mediated release of cytochrome c from mitochondria, and the mitochondrial electrochemical potential was only partly reduced. These results argue that BAP31 cleavage is important for manifesting cytoplasmic apoptotic events associated with membrane fragmentation and reveal an unexpected cross talk between mitochondria and the endoplasmic reticulum during Fas-mediated apoptosis in vivo.     

BAP31 and its caspase cleavage product regulate cell surface expression of tetraspanins and integrin-mediated cell survival

BAP31, a resident integral protein of the endoplasmic reticulum membrane, regulates the export of other integral membrane proteins to the downstream secretory pathway. Here we show that cell surface expression of the tetraspanins CD9 and CD81 is compromised in mouse cells from which the Bap31 gene has been deleted. CD9 and CD81 facilitate the function of multiprotein complexes at the plasma membrane, including integrins. Of note, BAP31 does not appear to influence the egress of alpha5beta1 or alpha(v)beta3 integrins to the cell surface, but in Bap31-null mouse cells, these integrins are not able to maintain cellular adhesion to the extracellular matrix in the presence of reduced serum. Consequently, Bap31-null cells are sensitive to serum starvation-induced apoptosis. Reconstitution of wild-type BAP31 into these Bap31-null cells restores integrin-mediated cell attachment and cell survival after serum stress, whereas interference with the functions of CD9, alpha5beta1, or alpha(v)beta3 by antagonizing antibodies makes BAP31 cells act similar to Bap31-null cells in these respects. Finally, in human KB epithelial cells protected from apoptosis by BCL-2, the caspase-8 cleavage product, p20 BAP31, inhibits egress of tetraspanin and integrin-mediated cell attachment. Thus, p20 BAP31 can operate upstream of BCL-2 in living cells to influence cell surface properties due to its effects on protein egress from the endoplasmic reticulum.     

Epitope Mapping of Antibodies Suggests the Novel Membrane Topology of B-Cell Receptor Associated Protein 31 on the Cell Surface of Embryonic Stem Cells: The Novel Membrane Topology of BAP31

When located in the endoplasmic reticulum (ER) membrane, B-cell receptor associated protein 31 (BAP31) is involved in the export of secreted proteins from the ER to the plasma membrane. In a previous study, we generated two monoclonal antibodies (mAbs), 297-D4 and 144-A8, that bound to surface molecules on human embryonic stem cells (hESCs), but not to surface molecules on mouse embryonic stem cells (mESCs). Subsequent studies revealed that the mAbs recognized BAP31 on the surface of hESCs. To investigate the membrane topology of BAP31 on the cell surface, we first examined the epitope specificity of 297-D4 and 144-A8, as well as a polyclonal anti-BAP31 antibody (α-BAP31). We generated a series of GST-fused BAP31 mutant proteins in which BAP31 was serially deleted at the C- terminus. GST-fused BAP31 mutant proteins were then screened to identify the epitopes targeted by the antibodies. Both 297-D4 and 144-A8 recognized C-terminal residues 208–217, while α-BAP31 recognized C-terminal residues 165–246, of BAP31 on hESCs, suggesting that the C-terminal domain of BAP31 is exposed on the cell surface. The polyclonal antibody α-BAP31 bound to mESCs, which confirmed that the C-terminal domain of BAP31 is also exposed on the surface of these cells. Our results show for the first time the novel membrane topology of cell surface-expressed BAP31 as the extracellular exposure of the BAP31 C-terminal domain was not predicted from previous studies.     

Uncleaved BAP31 in association with A4 protein at the endoplasmic reticulum is an inhibitor of Fas-initiated release of cytochrome c from mitochondria

BAP31 is a polytopic integral protein of the endoplasmic reticulum membrane and, like BID, is a preferred substrate of caspase-8. Upon Fas/CD95 stimulation, BAP31 is cleaved within its cytosolic domain, generating proapoptotic p20 BAP31. In human KB epithelial cells expressing the caspase-resistant mutant crBAP31, Fas stimulation resulted in cleavage of BID and insertion of BAX into mitochondrial membrane, but subsequent oligomerization of BAX and BAK, egress of cytochrome c to the cytosol, and apoptosis were impaired. Bap31-null mouse cells expressing crBAP31 cannot generate the endogenous p20 BAP31 cleavage product, yet crBAP31 conferred resistance to cellular condensation and cytochrome c release in response to activation of ectopic FKBPcasp8 by FK1012z. Full-length BAP31, therefore, is a direct inhibitor of these caspase-8-initiated events, acting independently of its ability to sequester p20, with which it interacts. Employing a novel split ubiquitin yeast two-hybrid screen for BAP31-interacting membrane proteins, the putative ion channel protein of the endoplasmic reticulum, A4, was detected and identified as a constitutive binding partner of BAP31 in human cells. Ectopic A4 that was introduced into A4-deficient cells cooperated with crBAP31 to resist Fas-induced egress of cytochrome c from mitochondria and cytoplasmic apoptosis.     

Inhibition of BAP31 expression inhibits cervical cancer progression by suppressing metastasis and inducing intrinsic and extrinsic apoptosis

Cervical cancer is reported as one of the most lethal types of cancer among female. However, extensive studies of the molecular mechanisms that regulate the progression of cervical cancer are still required. B-cell associated protein (BAP)-31 is a 28-kDa integral membrane protein in the endoplasmic reticulum (ER), playing essential role in modulating various physiological processes. The present study indicated that BAP31 was a novel gene associated with cervical cancer development. Here, we demonstrated that BAP31 was significantly increased in human cervical cancer specimens, which was positively correlated to histological grade of the cancer. BAP31 knockdown suppressed cell proliferation, clonogenic ability and metastasis-associated traits in vitro, as well as carcinogenesis and pulmonary metastasis in vivo. Further studies indicated that the expression levels of transforming growth factor (TGF)-β1, matrix metalloproteinase (MMP)-2, MMP-9, Rho-associated protein kinase 1 (ROCK1), α-smooth muscle actin (α-SMA), Vimentin and N-cadherin were markedly reduced by BAP31 knockdown in cervical cancer cells. In addition, intrinsic and extrinsic apoptosis was significantly induced in BAP31 knockdown cells, as evidenced by the increased expression of cleaved Caspase-8/-9/-3 and poly (ADP-ribose) polymerases (PARP). Notably, suppressing the activities of Caspase-8/-9 and ?3 obviously diminished BAP31 silence-triggered apoptosis. Together, these findings highlighted an essential role for BAP31 in the modulation of tumorigenesis and metastatic potential of cervical cancer, and demonstrated a promising application of BAP31 in cancer prevention.     

BAP31 interacts with Sec61 translocons and promotes retrotranslocation of CFTRDeltaF508 via the derlin-1 complex

BAP31 is an endoplasmic reticulum protein-sorting factor that associates with newly synthesized integral membrane proteins and controls their fate (i.e., egress, retention, survival, or degradation). BAP31 is itself an integral membrane protein and a constituent of several large protein complexes. Here, we show that a part of the BAP31 population interacts with two components of the Sec61 preprotein translocon, Sec61beta and TRAM. BAP31 associates with the N terminus of one of its newly synthesized client proteins, the DeltaF508 mutant of CFTR, and promotes its retrotranslocation from the ER and degradation by the cytoplasmic 26S proteasome system. Depletion of BAP31 reduces the proteasomal degradation of DeltaF508 and permits a significant fraction of the surviving protein to reach the cell surface. Of note, BAP31 also associates physically and functionally with the Derlin-1 protein disclocation complex in the DeltaF508 degradation pathway. Thus, BAP31 operates at early steps to deliver newly synthesized CFTRDeltaF508 to its degradation pathway.     

Association of BAP31 with CD11b/CD18. Potential role in intracellular trafficking of CD11b/CD18 in neutrophils

The beta2 integrin CD11b/CD18 is an integral membrane protein that is present in the plasma membrane and secondary granules of neutrophils and functions as a major adhesion molecule. Upon cellular activation, there is translocation of intracellular pools of CD11b/CD18 to the plasma membrane in concert with enhanced cellular adhesion. Although much is known about the function of CD11b/CD18, how this protein is transported within the cell is less well defined. Here we report that CD11b/CD18 specifically binds to BAP31, a member of a novel class of sorting proteins regulating cellular anterograde transport. Through experiments aimed at identifying CD11b/CD18-binding proteins, we produced a monoclonal antibody termed E1B2 that recognizes a 28-kDa membrane protein that co-precipitates with CD11b/CD18. Microsequence analysis of the E1B2 antigen revealed that it is BAP31. Co-association of CD11b/CD18 and BAP31 was confirmed in co-immunoprecipitation and protein binding assays. Additional experiments revealed that the binding of BAP31 to CD11b/CD18 was not dependent on divalent cations nor mediated by the I-domain of CD11b. Using glutathione S-transferase fusion chimeras, we determined that binding of CD11b/CD18 to BAP31 is mediated through interactions with the cytoplasmic tail of BAP31. Immunolocalization studies revealed colocalization of BAP31 and CD11b/CD18 within neutrophil secondary granules. Subcellular fractionation studies in polymorphonuclear leukocytes (PMN) revealed similar patterns of redistribution of BAP31 and CD11b/CD18 from fractions enriched in secondary granules to the plasma membrane following stimulation with formylmethionylleucylphenylalanine (fMLP). Given the known sorting properties of BAP31, these findings suggest that BAP31 may play a role in regulating intracellular trafficking of CD11b/CD18 in neutrophils.     

The yeast split-ubiquitin membrane protein two-hybrid screen identifies BAP31 as a regulator of the turnover of endoplasmic reticulum-associated protein tyrosine phosphatase-like B

In the past decade, traditional yeast two-hybrid techniques have identified a plethora of interactions among soluble proteins operating within diverse cellular pathways. The discovery of associations between membrane proteins by genetic approaches, on the other hand, is less well established due to technical limitations. Recently, a split-ubiquitin system was developed to overcome this barrier, but so far, this system has been limited to the analysis of known membrane protein interactions. Here, we constructed unique split-ubiquitin-linked cDNA libraries and provide details for implementing this system to screen for binding partners of a bait protein, in this case BAP31. BAP31 is a resident integral protein of the endoplasmic reticulum, where it operates as a chaperone or cargo receptor and regulator of apoptosis. Here we describe a novel human member of the protein tyrosine phosphatase-like B (PTPLB) family, an integral protein of the endoplasmic reticulum membrane with four membrane-spanning alpha helices, as a BAP31-interacting protein. PTPLB turns over rapidly through degradation by the proteasome system. Comparisons of mouse cells with a deletion of Bap31 or reconstituted with human BAP31 indicate that BAP31 is required to maintain PTPLB, consistent with a chaperone or quality control function for BAP31 in the endoplasmic reticulum membrane.     

Mutations in BCAP31 Cause a Severe X-Linked Phenotype with Deafness,Dystonia, and Central Hypomyelination and Disorganize the Golgi Apparatus

BAP31 is one of the most abundant endoplasmic reticulum (ER) membrane proteins. It is a chaperone protein involved in several pathways, including ER-associated degradation, export of ER proteins to the Golgi apparatus, and programmed cell death. BAP31 is encoded by BCAP31, located in human Xq28 and highly expressed in neurons. We identified loss-of-function mutations in BCAP31 in seven individuals from three families. These persons suffered from motor and intellectual disabilities, dystonia, sensorineural deafness, and white-matter changes, which together define an X-linked syndrome. In the primary fibroblasts of affected individuals, we found that BCAP31 deficiency altered ER morphology and caused a disorganization of the Golgi apparatus in a significant proportion of cells. Contrary to what has been described with transient-RNA-interference experiments, we demonstrate that constitutive BCAP31 deficiency does not activate the unfolded protein response or cell-death effectors. Rather, our data demonstrate that the lack of BAP31 disturbs ER metabolism and impacts the Golgi apparatus, highlighting an important role for BAP31 in ER-to-Golgi crosstalk. These findings provide a molecular basis for a Mendelian syndrome and link intracellular protein trafficking to severe congenital brain dysfunction and deafness.     

Endoplasmic reticulum membrane-sorting protein of lymphocytes (BAP31) is highly expressed in neurons and discrete endocrine cells.

BAP31 is a transmembrane protein that associates with nascent membrane proteins in transit between endoplasmic reticulum (ER) and cis-Golgi. Its C-terminal dilysine (KKEE) motif, mediating return to the ER, is consistent with a role in early sorting of membrane proteins. An initiator caspase-binding site in the C-terminal domain of BAP31 is implicated in cytoplasmic membrane fragmentation events of apoptosis. Although BAP31 RNA is ubiquitous, the protein's anatomic localization has not been determined. To gain further insight into its possible functions, we localized BAP31 in primate tissues using monoclonal antibodies. Immunoreactivity was prominent in T- and B-lymphocytes in blood and in thymus, in cerebellar Purkinje neuron bodies and dendrites, in gonadotrophs of the anterior pituitary, ovarian thecal and follicular cells, active but not quiescent thyroid epithelium, adrenal cortex more than medulla, and proximal more than distal renal tubules. Blood vessels and skeletal muscle were nonreactive. The anatomic distribution of BAP31 and the nature of proteins identified thus far as its cargo exiting the ER, suggest an interaction with proteins assembling in macromolecular complexes en route to selected sites of exocytotic and signaling activities. Apoptotic associations in mature tissues could be physiological (lymphocytes, endocrine cells) or pathological (Purkinje neurons, renal tubules).     

Limited caspase cleavage of human BAP31

Human BAP31 was cleaved at both of its two identical caspase cleavage sites in two previously reported models of apoptosis. We show here that only the most carboxy-terminal site is cleaved during apoptosis induced in HeLa cells by tunicamycin, tumor necrosis factor and cycloheximide, or staurosporine. Similar results were obtained in HL-60 cells using Fas/APO-1 antibodies, or cycloheximide. This limited cleavage, which is inhibited by several caspase inhibitors, removes eight amino acids from human BAP31 including the KKXX coat protein I binding motif. Ectopic expression of the resulting cleavage product induces redistribution of mannosidase II from the Golgi and prevents endoplasmic reticulum to Golgi transport of virus glycoproteins.     

The specificity of association of the IgD molecule with the accessory proteins BAP31/BAP29 lies in the IgD transmembrane sequence.

Mature B cells co-express on their cell surface two classes of antigen receptor, the IgM and IgD immunoglobulins. The structural and functional differences between the two receptor classes are poorly understood. Recently two proteins of 29 and 31 kDa (BAP29 and BAP31) have been described that are preferentially associated with membrane IgD but only weakly with membrane IgM. We describe here the cloning of full-length murine and human BAP31 cDNAs encoding proteins of 245 and 246 amino acids respectively. The two BAP31 proteins are 95% identical. The BAP31 gene is ubiquitously expressed in murine tissues and is located on the X chromosome in both mouse and man. The murine BAP31 protein has 43% sequence identity to murine BAP29. Both proteins have a hydrophobic N-terminus and an alpha-helical C-terminus which ends with a KKXX motif implicated in vesicular transport. By a mutational analysis we have identified amino acids in the transmembrane sequence of the delta m chain that are critical for binding to BAP31/BAP29. A structural model of the BAPs and their potential functions are discussed.     

Export of Cellubrevin from the Endoplasmic Reticulum Is Controlled by BAP31

Cellubrevin is a ubiquitously expressed membrane protein that is localized to endosomes throughout the endocytotic pathway and functions in constitutive exocytosis. We report that cellubrevin binds with high specificity to BAP31, a representative of a highly conserved family of integral membrane proteins that has recently been discovered to be binding proteins of membrane immunoglobulins. The interaction between BAP31 and cellubrevin is sensitive to high ionic strength and appears to require the transmembrane regions of both proteins. No other proteins of liver membrane extracts copurified with BAP31 on immobilized recombinant cellubrevin, demonstrating that the interaction is specific. Synaptobrevin I bound to BAP31 with comparable affinity, whereas only weak binding was detectable with synaptobrevin II. Furthermore, a fraction of BAP31 and cellubrevin was complexed when each of them was quantitatively immunoprecipitated from detergent extracts of fibroblasts (BHK 21 cells). During purification of clathrin-coated vesicles or early endosomes, BAP31 did not cofractionate with cellubrevin. Rather, the protein was enriched in ER-containing fractions. When BHK cells were analyzed by immunocytochemistry, BAP31 did not overlap with cellubrevin, but rather colocalized with resident proteins of the ER. In addition, immunoreactive vesicles were clustered in a paranuclear region close to the microtubule organizing center, but different from the Golgi apparatus. When microtubules were depolymerized with nocodazole, this accumulation disappeared and BAP31 was confined to the ER. Truncation of the cytoplasmic tail of BAP31 prevented export of cellubrevin, but not of the transferrin receptor from the ER. We conclude that BAP31 represents a novel class of sorting proteins that controls anterograde transport of certain membrane proteins from the ER to the Golgi complex.Exocytotic membrane fusion is mediated by a complex of evolutionary-conserved membrane proteins. In neurons, these proteins include the synaptic vesicle protein synaptobrevin (VAMP) and the synaptic membrane proteins syntaxin and synaptosome-associated protein (SNAP)-25.¹ These proteins undergo regulated protein–protein interactions that are controlled by soluble proteins including N-ethylmaleimide-sensitive factor (NSF) and soluble N-ethyl maleimide-sensitive factor attachment (SNAP) proteins (Söllner et al., 1993b ). Relatives of all of these proteins have been discovered in many eukaryotic cells including yeast, suggesting that intracellular membrane fusions may, at least to a large extent, be mediated by common mechanisms (Ferro-Novick and Jahn, 1994; Rothman, 1994; Scheller, 1995). Although the molecular details of membrane fusion are not yet understood, it is becoming clear that the components of the fusion apparatus operate by conformation-dependent assembly and disassembly reactions which ultimately lead to the rearrangement of membrane phospholipids (Söllner et al., 1993a ; Calakos et al., 1994). For these reasons, the interactions between synaptobrevin, SNAP-25, and syntaxin have received considerable attention (for review see Südhof, 1995). These proteins form a tight and stable ternary complex as soon as they have access to each other. Binding probably occurs before or during vesicle docking in preparation for fusion. Incubation with the ATPase NSF and SNAP proteins reversibly disassembles this complex, an event thought to precede membrane fusion (Söllner et al., 1993a ,b).It is less well understood to what extent synaptobrevin, SNAP-25, and syntaxin interact with other proteins, particularly during stages of their life cycle when they are not bound to each other. It is conceivable that companion proteins exist that assist in sorting to the correct compartment or in positioning at the site of release and that control the availability for entering the fusion complex. For syntaxin, interactions with several other proteins were reported, including synaptotagmin munc-18/rbSEC-1, and the N-type Ca²⁺-channel (Südhof, 1995). For synaptobrevin, it has recently been observed that most of the protein is associated with synaptophysin, an integral membrane protein of yet unknown function that resides alongside synaptobrevin in the synaptic vesicle membrane (Calakos and Scheller, 1994; Edelmann et al., 1995; Washbourne et al., 1995). Although the binary interaction of synaptobrevin with synaptophysin is weaker than its ternary interaction with syntaxin and SNAP-25, synaptophysin-bound synaptobrevin is not available for binding to these proteins (Edelmann et al., 1995). Thus, synaptobrevin participates at least in two different complexes that are mutually exclusive: one with its partners syntaxin and SNAP-25 during membrane fusion, and another with synaptophysin during vesicle recycling and probably also during biogenesis, i.e., during transport of the proteins from the ER to the nerve terminal.It remains to be established whether cellubrevin, a nonneuronal synaptobrevin homologue with widespread distribution, forms partnerships with other proteins with properties similar to the synaptobrevin–synaptophysin complex. Like synaptobrevins, cellubrevin is a small integral membrane protein with a single transmembrane domain at the COOH-terminal end of the molecule. Cellubrevin colocalizes with the transferrin receptor in fibroblasts and is enriched in purified clathrin-coated vesicles (McMahon et al., 1993), suggesting that it resides in constitutive trafficking vesicles shuttling mainly between the plasmalemma and the endosomal compartment (Daro et al., 1996). Like its neuronal counterparts, cellubrevin is selectively cleaved by clostridial neurotoxins including tetanus toxin. Toxin cleavage impairs exocytosis of recycling vesicles in fibroblasts (Galli et al., 1994), whereas fusion of early endosomes appears not to be affected (Link et al., 1993; Jo et al., 1995).Here we report that cellubrevin interacts specifically with a recently characterized integral membrane protein, BAP31. BAP31 and a related protein (BAP29) were first identified as membrane proteins copurifying with membrane-bound immunoglobulin from lysates of β lymphocytes (Kim et al., 1994). Cloning of human and murine BAP31 cDNA showed that BAP31 is an evolutionary-conserved protein which is ubiquitously expressed in all tissues (Adachi et al., 1996). Several open reading frames encoding for proteins with a similar structure and a significant degree of homology are present in the genome of the yeast Saccharomyces cerevisiae, suggesting that BAP31 represents an ancient protein family with basic functions (EMBL/GenBank/DDBJ accession numbers {"type":"entrez-nucleotide","attrs":{"text":"Z28065","term_id":"486088","term_text":"Z28065"}}Z28065, {"type":"entrez-nucleotide","attrs":{"text":"Z74120","term_id":"1431083","term_text":"Z74120"}}Z74120, and {"type":"entrez-nucleotide","attrs":{"text":"Z48502","term_id":"695715","term_text":"Z48502"}}Z48502). BAP31 has a hydrophobic NH₂ terminus with three potential transmembrane domains and a charged α-helical COOH terminus that is exposed to the cytoplasm. The COOH terminus ends with a KKXX sequence motif typical for proteins transported back to the ER. Indeed, an immunocytochemical analysis revealed that BAP31 exhibits an ER-like staining pattern (Becker, B., and M. Reth, unpublished observations). We show that BAP31, as a resident of the ER and of ER-derived trafficking vesicles, may control the export of cellubrevin from the ER.     

Rescue of CH31 B cells from antigen receptor-induced apoptosis by inhibition of p38 MAPK

CH31 B lymphomas represent a model for antigen-induced deletional tolerance of immature B lymphocytes, because cross-linking the B cell antigen receptor (BCR) induces G(1) phase arrest and apoptosis. We have recently demonstrated that BCR cross-linking leads to a transient activation of p38 mitogen-activated protein kinase (MAPK) in CH31 B cells. In this paper, we functionally characterize the role of p38 MAPK in BCR-induced apoptosis as well as evaluate the regulation of additional MAPKs by the BCR. We demonstrate that JNK and ERK activities are not affected by BCR cross-linking, suggesting that these MAPKs are not directly involved in initiating the apoptotic cascade. By contrast, we show that pretreatment of CH31 B cells with the highly specific p38 MAPK inhibitor SB203580 ablated both BCR-induced p38 MAPK activity and apoptosis. Pretreatment of CH31 cells with an inactive SB203580 analog, SB202474, did not prevent apoptosis. These findings establish a key role for p38 MAPK in antigen receptor-mediated apoptosis of CH31 B cells.     

Nonmuscle myosin II is required for cell proliferation, cell sheet adhesion and wing hair morphology during wing morphogenesis

Metazoan development involves a myriad of dynamic cellular processes that require cytoskeletal function. Nonmuscle myosin II plays essential roles in embryonic development; however, knowledge of its role in post-embryonic development, even in model organisms such as Drosophila melanogaster, is only recently being revealed. In this study, truncation alleles were generated and enable the conditional perturbation, in a graded fashion, of nonmuscle myosin II function. During wing development they demonstrate novel roles for nonmuscle myosin II, including in adhesion between the dorsal and ventral wing epithelial sheets; in the formation of a single actin-based wing hair from the distal vertex of each cell; in forming unbranched wing hairs; and in the correct positioning of veins and crossveins. Many of these phenotypes overlap with those observed when clonal mosaic analysis was performed in the wing using loss of function alleles. Additional requirements for nonmuscle myosin II are in the correct formation of other actin-based cellular protrusions (microchaetae and macrochaetae). We confirm and extend genetic interaction studies to show that nonmuscle myosin II and an unconventional myosin, encoded by crinkled (ck/MyoVIIA), act antagonistically in multiple processes necessary for wing development. Lastly, we demonstrate that truncation alleles can perturb nonmuscle myosin II function via two distinct mechanisms—by titrating light chains away from endogenous heavy chains or by recruiting endogenous heavy chains into intracellular aggregates. By allowing myosin II function to be perturbed in a controlled manner, these novel tools enable the elucidation of post-embryonic roles for nonmuscle myosin II during targeted stages of fly development.     

HIV-1 Vpr-induced apoptosis is cell cycle dependent and requires Bax but not ANT

The HIV-1 accessory protein viral protein R (Vpr) causes G2 arrest and apoptosis in infected cells. We previously identified the DNA damage-signaling protein ATR as the cellular factor that mediates Vpr-induced G2 arrest and apoptosis. Here, we examine the mechanism of induction of apoptosis by Vpr and how it relates to induction of G2 arrest. We find that entry into G2 is a requirement for Vpr to induce apoptosis. We investigated the role of the mitochondrial permeability transition pore by knockdown of its essential component, the adenine nucleotide translocator. We found that Vpr-induced apoptosis was unaffected by knockdown of ANT. Instead, apoptosis is triggered through a different mitochondrial pore protein, Bax. In support of the idea that checkpoint activation and apoptosis induction are functionally linked, we show that Bax activation by Vpr was ablated when ATR or GADD45alpha was knocked down. Certain mutants of Vpr, such as R77Q and I74A, identified in long-term nonprogressors, have been proposed to inefficiently induce apoptosis while activating the G2 checkpoint in a normal manner. We tested the in vitro phenotypes of these mutants and found that their abilities to induce apoptosis and G2 arrest are indistinguishable from those of HIV-1NL4-3 vpr, providing additional support to the idea that G2 arrest and apoptosis induction are mechanistically linked.