The resident endoplasmic reticulum protein, BAP31, associates with gamma-actin and myosin B heavy chain.

BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two caspase recognition sites that are preferentially cleaved by initiator caspases, such as caspase-8. Recently, we reported that the caspase-resistant BAP31 inhibited Fas-mediated apoptotic membrane fragmentation and the release of cytochrome c from mitochondria in KB epithelial cells (Nguyen M., Breckenridge G., Ducret A & Shore G. (2000) Mol. Cell. Biol.20, 6731-6740). We describe here the characterization by capillary liquid chromatography microelectrospray tandem MS of a BAP31 immunocomplex isolated from a HepG2 cell lysate in the absence of a death signal. We show that BAP31 specifically associates with nonmuscle myosin heavy chain B and nonmuscle gamma-actin, two components of the cytoskeleton actomyosin complex. Collectively, these data confirm that BAP31, in addition to its potential role as a chaperone, may play a fundamental role in the structural organization of the cytoplasm. Here we also show that Fas stimulation of apoptosis releases BAP31 associations with these motor proteins, a step that may contribute to extranuclear events, such as membrane remodelling, during the execution phase of apoptosis.     

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.     

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.     

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.     

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.     

Mammalian BiP controls posttranslational ER translocation of the hepatitis B virus large envelope protein

The hepatitis B virus L protein forms a dual topology in the endoplasmic reticulum (ER) via a process involving cotranslational membrane integration and subsequent posttranslational translocation of its preS subdomain. Here, we show that preS posttranslocation depends on the action of the ER chaperone BiP. To modulate the in vivo BiP activity, we designed an approach based on overexpressing its positive and negative regulators, ER-localized DnaJ-domain containing protein 4 (ERdj4) and BiP-associated protein (BAP), respectively. The feasibility of this approach was confirmed by demonstrating that BAP, but not ERdj4, destabilizes the L/BiP complex. Overexpressing BAP or ERdj4 inhibits preS posttranslocation as does the reduction of ATP levels. These results hint to a new role of BiP in guiding posttranslational polypeptide import into the mammalian ER.     

TPR-containing proteins control protein organization and homeostasis for the endoplasmic reticulum

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into a number of functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein–protein interactions, supporting interactions with a wide range of ligands or substrates. Seven TPR-containing proteins have thus far been shown to localize to the ER and control protein organization and homeostasis within this multifunctional organelle. Here, we discuss the roles of these proteins in controlling ER processes and organization. The crucial roles that TPR-containing proteins play in the ER are highlighted by diseases or defects associated with their mutation or disruption.     

BAP31 and BiP are essential for dislocation of SV40 from the endoplasmic reticulum to the cytosol

How non-enveloped viruses overcome host cell membranes is poorly understood. Here, we show that after endocytosis and transport to the endoplasmic reticulum (ER), but before crossing the ER membrane to the cytosol, incoming simian virus 40 particles are structurally remodelled leading to exposure of the amino-terminal sequence of the minor viral protein VP2. These hydrophobic sequences anchor the virus to membranes. A negatively charged residue, Glu 17, in the α-helical, membrane-embedded peptide is essential for infection, most likely by introducing an 'irregularity' recognized by the ER-associated degradation (ERAD) system for membrane proteins. Using a siRNA-mediated screen, the lumenal chaperone BiP and the ER-membrane protein BAP31 (both involved in ERAD) were identified as being essential for infection. They co-localized with the virus in discrete foci and promoted its ER-to-cytosol dislocation. Virus-like particles devoid of VP2 failed to cross the membrane. The results demonstrated that ERAD-factors assist virus transport across the ER membrane.     

Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress

Hyperhomocysteinemia, a risk factor for vascular disease, injures endothelial cells through undefined mechanisms. We previously identified several homocysteine-responsive genes in cultured human vascular endothelial cells, including the endoplasmic reticulum (ER)-resident molecular chaperone GRP78/BiP. Here, we demonstrate that homocysteine induces the ER stress response and leads to the expression of a novel protein, Herp, containing a ubiquitin-like domain at the N terminus. mRNA expression of Herp was strongly up-regulated by inducers of ER stress, including mercaptoethanol, tunicamycin, A23187, and thapsigargin. The ER stress-dependent induction of Herp was also observed at the protein level. Immunochemical analyses using Herp-specific antibodies indicated that Herp is a 54-kDa, membrane-associated ER protein. Herp is the first integral membrane protein regulated by the ER stress response pathway. Both the N and C termini face the cytoplasmic side of the ER; this membrane topology makes it unlikely that Herp acts as a molecular chaperone for proteins in the ER, in contrast to GRP78 and other ER stress-responsive proteins. Herp may, therefore, play an unknown role in the cellular survival response to stress.     

Control of cystic fibrosis transmembrane conductance regulator expression by BAP31

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is stringently controlled by molecular chaperones participating in formation of the quality control system. It has been shown that about 75% of all CFTR protein and close to 100% of the [DeltaPhe(508)] CFTR variant are rapidly degraded before leaving the endoplasmic reticulum (ER). B cell antigen receptor-associated proteins (BAPs) are ubiquitously expressed integral membrane proteins that may control association with the cytoskeleton, vesicular transport, or retrograde transport from the cis Golgi to the ER. The present study delivers evidence for cytosolic co-localization of both BAP31 and CFTR and for the control of expression of recombinant CFTR in Chinese hamster ovary (CHO) cells and Xenopus oocytes by BAP31. Antisense inhibition of BAP31 in various cell types increased expression of both wild-type CFTR and [DeltaPhe(508)]CFTR and enabled cAMP-activated Cl(-) currents in [DeltaPhe(508)]CFTR-expressing CHO cells. Coexpression of CFTR together with BAP31 attenuated cAMP-activated Cl(-) currents in Xenopus oocytes. These data therefore suggest association of BAP31 with CFTR that may control maturation or trafficking of CFTR and thus expression in the plasma membrane.     

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).     

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.     

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.     

Substrate relay in an Hsp70‐cochaperone cascade safeguards tail‐anchored membrane protein targeting

Membrane proteins are aggregation‐prone in aqueous environments, and their biogenesis poses acute challenges to cellular protein homeostasis. How the chaperone network effectively protects integral membrane proteins during their post‐translational targeting is not well understood. Here, biochemical reconstitutions showed that the yeast cytosolic Hsp70 is responsible for capturing newly synthesized tail‐anchored membrane proteins (TAs) in the soluble form. Moreover, direct interaction of Hsp70 with the cochaperone Sgt2 initiates a sequential series of TA relays to the dedicated TA targeting factor Get3. In contrast to direct loading of TAs to downstream chaperones, stepwise substrate loading via Hsp70 maintains the solubility and targeting competence of TAs, ensuring their efficient delivery to the endoplasmic reticulum (ER). Inactivation of cytosolic Hsp70 severely impairs TA translocation in vivo. Our results demonstrate a new role of cytosolic Hsp70 in directly assisting the targeting of an essential class of integral membrane proteins and provide a paradigm for how “substrate funneling” through a chaperone cascade preserves the conformational quality of nascent membrane proteins during their biogenesis.     

Relationship between calnexin and BiP in suppressing aggregation and promoting refolding of protein and glycoprotein substrates

Calnexin (CNX) is a membrane protein of the endoplasmic reticulum that has been defined primarily as a lectin, yet is capable of functioning as a molecular chaperone with non-glycosylated proteins in vitro. Here, we assess the relative contributions of the oligosaccharide- and polypeptide-binding sites of CNX to its in vitro chaperone functions by comparing it with the Hsp70 chaperone of the endoplasmic reticulum, BiP. Both proteins were equally effective in preventing the aggregation of non-glycosylated citrate synthase, indicating that the polypeptide-binding site of CNX is capable of functioning at a level similar to that of Hsp70. However, when confronted with glycoprotein substrates, the lectin site of CNX provided a significant advantage over BiP in suppressing aggregation. CNX also cooperated with BiP and the J domain of Sec63p in the ATP-dependent refolding of glycoprotein and non-glycosylated substrates. The lectin site of CNX was essential for refolding of the glycoprotein. These findings reinforce the function of CNX as a bona fide chaperone and illustrate how its lectin site confers advantages relative to other chaperones when confronted with glycoprotein substrates.     

Intracellular trafficking of KA2 kainate receptors mediated by interactions with coatomer protein complex I (COPI) and 14-3-3 chaperone systems

Assembly and trafficking of neurotransmitter receptors are processes contingent upon interactions between intracellular chaperone systems and discrete determinants in the receptor proteins. Kainate receptor subunits, which form ionotropic glutamate receptors with diverse roles in the central nervous system, contain a variety of trafficking determinants that promote either membrane expression or intracellular sequestration. In this report, we identify the coatomer protein complex I (COPI) vesicle coat as a critical mechanism for retention of the kainate receptor subunit KA2 in the endoplasmic reticulum. COPI subunits immunoprecipitated with KA2 subunits from both cerebellum and COS-7 cells, and beta-COP protein interacted directly with immobilized KA2 peptides containing the arginine-rich retention/retrieval determinant. Association between COPI proteins and KA2 subunits was significantly reduced upon alanine substitution of this signal in the cytoplasmic tail of KA2. Temperature-sensitive degradation of COPI complex proteins was correlated with an increase in plasma membrane localization of the homologous KA2 receptor. Assembly of heteromeric GluR6a/KA2 receptors markedly reduced association of KA2 and COPI. Finally, the reduction in COPI binding was correlated with an increased association with 14-3-3 proteins, which mediate forward trafficking of other integral signaling proteins. These interactions therefore represent a critical early checkpoint for biosynthesis of functional KARs.     

Assessment of the integral membrane protein topology in living cells

The bimolecular fluorescence complementation (BiFC) phenomenon has been successfully applied for in vivo protein-protein interaction studies and protein tagging analysis. Here we report a novel BiFC-based technique for investigation of integral membrane protein topology in living plant cells. This technique relies on the formation of a fluorescent complex between a non-fluorescent fragment of the yellow fluorescent protein (YFP) targeted into a specific cellular compartment and a counterpart fragment attached to the integral membrane protein N- or C-terminus or inserted into the internal loop(s). We employed this technique for topological studies of beet yellows virus-encoded p6 membrane-embedded movement protein, a protein with known topology, and the potato mop-top virus-encoded integral membrane TGBp2 protein with predicted topology. The results confirm that p6 is a type III integral transmembrane protein. Using a novel method, the central hydrophilic region of TGBp2 was localized into the ER lumen, whereas the N- and C-termini localized to the cytosol. We conclude that the BiFC-based reporter system for membrane protein topology analysis is a relatively fast and efficient method that can be used for high-throughput analysis of proteins integrated into the endoplasmic reticulum in living plant cells.     

Chaperone insufficiency links TLR4 protein signaling to endoplasmic reticulum stress

Inflammation plays an important pathogenic role in a number of metabolic diseases such as obesity, type 2 diabetes, and atherosclerosis. The activation of inflammation in these diseases depends at least in part on the combined actions of TLR4 signaling and endoplasmic reticulum stress, which by acting in concert can boost the inflammatory response. Defining the mechanisms involved in this phenomenon may unveil potential targets for the treatment of metabolic/inflammatory diseases. Here we used LPS to induce endoplasmic reticulum stress in the human monocyte cell-line, THP-1. The unfolded protein response, produced after LPS, was dependent on CD14 activity but not on RNA-dependent protein kinase and could be inhibited by an exogenous chemical chaperone. The induction of the endoplasmic reticulum resident chaperones, GRP94 and GRP78, by LPS was of a much lower magnitude than the effect of LPS on TLR4 and MD-2 expression. In face of this apparent insufficiency of chaperone expression, we induced the expression of GRP94 and GRP78 by glucose deprivation. This approach completely reverted endoplasmic reticulum stress. The inhibition of either GRP94 or GRP78 with siRNA was sufficient to rescue the protective effect of glucose deprivation on LPS-induced endoplasmic reticulum stress. Thus, insufficient LPS-induced chaperone expression links TLR4 signaling to endoplasmic reticulum stress.     

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.     

Membrane protein integration into the endoplasmic reticulum

Most integral membrane proteins are targeted, inserted and assembled in the endoplasmic reticulum membrane. The sequential and potentially overlapping events necessary for membrane protein integration take place at sites termed translocons, which comprise a specific set of membrane proteins acting in concert with ribosomes and, probably, molecular chaperones to ensure the success of the whole process. In this minireview, we summarize our current understanding of helical membrane protein integration at the endoplasmic reticulum, and highlight specific characteristics that affect the biogenesis of multispanning membrane proteins.