Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membrane-inserted diphtheria toxin T domain

The membrane topography of proteins that convert between soluble and membrane-inserted states has proven a challenging problem. In particular, it has been difficult to define both whether a transmembrane orientation is achieved and what are the boundaries of membrane-inserted segments. In this report the fluorescence of bimane-labeled Cys residues and the binding of anti-BODIPY antibodies to BODIPY-labeled Cys residues are combined to define these features for helices TH8 and TH9 of the T domain of diphtheria toxin. Using a series of labeled residues the topography of these helices was examined in both conformations of membrane-inserted T domain identified previously (Wang, Y., Malenbaum, S. E., Kachel, K., Zhan, H., Collier, R. J., and London, E. (1997) J. Biol. Chem. 272, 25091-25098). In the shallowly inserted conformation these helices are found to be aligned close to the cis surface of the bilayer all along their sequences. In contrast, in the more deeply inserted conformation most TH8 and TH9 residues examined located in a non-polar environment, with the boundaries of the membrane-inserted sequences close to residues 324 and 372-374 on the cis (insertion) side of the bilayer. It was also found that residues 348 and 349, which are in the loop connecting TH8 and TH9, reached the opposite trans side of the bilayer, but did not protrude fully into the aqueous environment. These boundaries suggest the membrane-inserted segments of TH8 and TH9 form transmembrane helices about 25 residues in length, and suggest that they are connected by a tight turn. It is concluded that this combination of fluorescent techniques can be combined to obtain transmembrane helix topography.     

The effects of helix breaking mutations in the diphtheria toxin transmembrane domain helix layers of the fusion toxin DAB389IL-2

Irritable bowel syndrome is frequently encountered in clinical practice, and it has been repeatedly suggested that abnormal colonic motor activity is one of the major pathophysiological mechanisms responsible for the origin of symptoms in such disorder. If this statement is true, then high-amplitude propagated colonic contractions (HAPCs), i.e. the mass movements, may play an important role. To test this hypothesis, we conducted an investigation by recording colonic motility for a prolonged (24 h) period in 25 patients with irritable bowel syndrome and in 18 healthy volunteers, to compare the number of mass movements over 24 h in patients (constipation-predominant, alternating bowel habits) and controls. The overall amount of motility was also assessed in twelve patients and 13 controls. We also looked for the possible changes in mass movements and motility which may occur with defecation and after a meal. The results showed that 1) with respect to HAPCs and motility index, neither group was significantly different from controls; 2) HAPCs and the motility index were significantly reduced during sleep in all groups tested; 3) HAPCs were significantly more common before as compared to after defecation and after as compared to before meals; 4) HAPCs are not independent from the segmental contractile activity; 5) the motility index/24 h was lower in the constipation-predominant group of patients with respect to controls. We conclude that in patients with irritable bowel syndrome colonic motility per se may play a pathophysiological role in the genesis of the symptoms, although other mechanisms are likely to concur, or to be responsible for the complaints of these patients. However, colonic prolonged recordings are very useful for studying physiological and pathophysiological correlates of sleep, eating, and defecation.     

Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: identification of the transmembrane domain as a site for oligomerization

Fatty acid amide hydrolase (FAAH) is an integral membrane protein responsible for the hydrolysis of a number of primary and secondary fatty acid amides, including the neuromodulatory compounds anandamide and oleamide. Analysis of FAAH's primary sequence reveals the presence of a single predicted transmembrane domain at the extreme N-terminus of the enzyme. A mutant form of the rat FAAH protein lacking this N-terminal transmembrane domain (DeltaTM-FAAH) was generated and, like wild type FAAH (WT-FAAH), was found to be tightly associated with membranes when expressed in COS-7 cells. Recombinant forms of WT- and DeltaTM-FAAH expressed and purified from Escherichia coli exhibited essentially identical enzymatic properties which were also similar to those of the native enzyme from rat liver. Analysis of the oligomerization states of WT- and DeltaTM-FAAH by chemical cross-linking, sedimentation velocity analytical ultracentrifugation, and size exclusion chromatography indicated that both enzymes were oligomeric when membrane-bound and after solubilization. However, WT-FAAH consistently behaved as a larger oligomer than DeltaTM-FAAH. Additionally, SDS-PAGE analysis of the recombinant proteins identified the presence of SDS-resistant oligomers for WT-FAAH, but not for DeltaTM-FAAH. Self-association through FAAH's transmembrane domain was further demonstrated by a FAAH transmembrane domain-GST fusion protein which formed SDS-resistant dimers and large oligomeric assemblies in solution.     

The membrane proximal cytokine receptor domain of the human interleukin-6 receptor is sufficient for ligand binding but not for gp130 association

Interleukin-6 (IL-6) belongs to the family of the "four-helix bundle" cytokines. The extracellular parts of their receptors consist of several Ig- and fibronectin type III-like domains. Characteristic of these receptors is a cytokine-binding module consisting of two such fibronectin domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine (WSXWS) sequence motif. On target cells, IL-6 binds to a specific IL-6 receptor (IL-6R), and the complex of IL-6.IL-6R associates with the signal transducing protein gp130. The IL-6R consists of three extracellular domains. The NH2-terminal Ig-like domain is not needed for ligand binding and signal initiation. Here we have investigated the properties and functional role of the third membrane proximal domain. The protein can be efficiently expressed in bacteria, and the refolded domain is shown to be sufficient for IL-6 binding. When complexed with IL-6, however, it fails to associate with the gp130 protein. Since the second and the third domain together with IL-6 can bind to gp130 and induce signaling, our data demonstrate the ligand binding function of the third domain and point to an important role of the second domain in complex formation with gp130 and signaling.     

Genetic construction and characterization of an anti-monkey CD3 single-chain immunotoxin with a truncated diphtheria toxin

In arteries, adrenomedullin (ADM) causes relaxations of rings with and without endothelium by stimulating accumulation of cyclic nucleotides resulting from activation of the ADM and calcitonin gene-related peptide (CGRP) receptors. Experiments were designed to determine the mechanism(s) of relaxation to ADM in veins. Rings of canine femoral vein with and without endothelium were suspended in organ chambers for measurement of isometric force. Rings were contracted with prostaglandin F2alpha (2 x 10(-6) M), and cumulative dose-responses to ADM (10(-11) to 10(-7) M) were obtained in the absence or presence of indomethacin (10(-5) M), indomethacin + N(G)-monomethyl-L-arginine (10(-4) M), methylene blue (10(-5) M), particulate guanylate cyclase inhibitor HS-142-1 (10(-5) M), tetraethylammonium (TEA, 10(-2) M), CGRP-receptor antagonist (CGRP 8-37, 10(-6) M), ADM-receptor antagonist (ADM 26-52, 10(-6) M), diphenhydramine (10(-6) M), 8-phenyltheophylline (3 x 10(-6) M), or superoxide dismutase (150 U/ml) plus catalase (1,200 U/ml). ADM produced concentration-dependent relaxations only in veins with endothelium. Relaxations to ADM in rings with endothelium were significantly inhibited only by methylene blue and HS-142-1. In separate experiments, incubation of rings with ADM (10(-8) M) and 3-isobutyl-1-methyl-xanthine (10(-4) M) for 3 min did not significantly affect the accumulation of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP). These data suggest that ADM-mediated relaxation in veins is endothelium dependent and is not associated with activation of CGRP receptors or currently defined ADM receptors. Further, relaxations are not mediated by nitric oxide, indomethacin-sensitive prostanoids, TEA-sensitive hyperpolarizing factors, oxygen free radicals, or accumulation of cyclic nucleotides.     

Immunotherapy for medullary thyroid carcinoma by a replication-defective adenovirus transducing murine interleukin-2

We have evaluated the feasibility of gene transduction using replication-defective adenovirus vector as a novel therapy for medullary thyroid carcinoma (MTC), a thyroid C cell neoplasm. Replication-defective adenoviruses were constructed to express murine interleukin-2 (mIL-2) gene and Escherichia coli beta-galactosidase (beta-gal; lacZ) gene under the control of the human cytomegalovirus (CMV) promoter (AdCMVmIL2, AdCMVbeta-gal) by homologous recombination. The efficiency of transduction was evaluated using AdCMVbeta-gal at different conditions. The gene transduction efficiency was dependent on multiplicity of infection, duration of exposure to the virus, and viral concentration. The expression of functional mIL-2 in transduced tumor cells was verified both in vitro and in vivo. Two cell lines (rat MTC and mMTC) secreted large amounts of functional mIL-2 after transduction, as tested in cytotoxic T lymphocyte (CTL) L-2 cells. When AdCMVmIL2-infected mMTC cells were injected s.c. into their host animals, tumors developed in 2 of 10 animals, in contrast to 9 of 10 animals injected with AdCMVbeta-gal-infected mMTC cells and all 10 animals injected with parental mMTC cells. Moreover protected animals developed a long lasting immunity against mMTC tumor cells and their splenocytes, showing cytotoxicity to parental tumor cells, and active natural killer (NK) cell activity. BALB/c-SCID (severe combined immune deficiency) mice were also used to evaluate the function of NK cells in antitumor activities. No tumor developed in SCID mice injected with AdCMVmIL2-infected cells, whereas all animals injected with either AdCMVbeta-gal-infected or parental mMTC cells developed tumors. Our data indicate that IL-2 production by MTC cells leads to rejection in syngeneic animals and suggest that both cytotoxic T cells and NK cells may play an important role. In addition, transduction of adenoviral vectors into tumor cells produces some nonspecific antitumor effects.     

Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity

The membrane anchored form of human heparin-binding epidermal growth factor-like growth factor (HB-EGF) acts as the diphtheria toxin (DT) receptor. Transfection of human HB-EGF cDNA into mouse LC cells, L cells stably expressing DRAP27, conferred sensitivity to DT, but transfection of mouse HB-EGF cDNA did not. To define the essential regions of HB-EGF that serve as the functional DT receptor, we examined the sensitivity to DT and DT binding of cells expressing several human/mouse HB-EGF chimeras. It was found that DT binds to the EGF-like domain of the human HB-EGF. However, mouse HB-EGF does not serve as a functional DT receptor due to non-conserved amino acid substitutions in this domain. In addition, CRM197, a non-toxic mutant of DT, inhibited strongly the mitogenic activity of the secreted form of human HB-EGF, but not of mouse HB-EGF and other EGF receptor-binding growth factors. These results confirmed further that DT interacts with the EGF-like domain of HB-EGF and that this interaction is specific for human HB-EGF.     

The antiviral action of recombinant receptorotoxins based on the diphtheria toxin and the human T-lymphocyte CD4-receptor

Lack of uniformity in the classification of oral diseases, and variability of study designs, measurements, methods, and statistical formats, hamper the interpretation, comparison and review of the evidence linking smoking and oral diseases. However, there have been a significant number of controlled studies, allowing definitive conclusions to be drawn. This review of 22 controlled scientific studies from 1983-1992, considers the role of smoking as an aetiological agent in: gingival problems (impaired gingival bleeding, ANUG), periodontal problems (periodontitis, bone loss, tooth loss), and caries.     

The cystic fibrosis transmembrane conductance regulator as a marker of human pancreatic duct development

BACKGROUND & AIMS: The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a small conductance adenosine 3',5'-cyclic monophosphate (cAMP)-activated chloride ion channel found in the apical membranes of epithelia within the pancreas, airway, intestine, bile duct, sweat gland, and male genital ducts. Pancreatic insufficiency is a feature of about 85% of patients with cystic fibrosis and is believed to be caused by pancreatic autolysis after pancreatic duct obstruction. The aim of this study was to investigate the expression of CFTR in the pancreas from early development to postnatal life to establish whether the CFTR plays a key role in development of the pancreatic duct epithelium. METHODS: Expression of CFTR from the start of the mid-trimester of human development through term to adult life by messenger RNA (mRNA) in situ hybridization was examined. RESULTS: CFTR mRNA is detected throughout the pancreatic duct epithelium and its pattern of expression follows the differentiation of the duct system. CONCLUSIONS: CFTR is a valuable marker of human pancreatic duct cell development and differentiation.     

Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion

Escherichia coli biotin ligase is a cytoplasmic protein which specifically biotinylates the biotin-accepting domains from a variety of organisms. This in vivo biotinylation can be used as a sensitive signal to study protein secretion and membrane protein insertion. When the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (PSBT) is translationally fused to the periplasmic proteins alkaline phosphatase and maltose-binding protein, there is little or no biotinylation of PSBT in wild-type E. coli. Inhibition of SecA with sodium azide and mutations in SecB, SecD, and SecF, all of which slow down protein secretion, result in biotinylation of PSBT. When PSBT is fused to the E. coli inner membrane protein MalF, it acts as a topological marker: fusions to cytoplasmic domains of MalF are biotinylated, and fusions to periplasmic domains are generally not biotinylated. If SecA is inhibited by sodium azide or if the SecE in the cell is depleted, then the insertion of the MalF second periplasmic domain is slowed down enough that PSBT fusions in this part of the protein become biotinylated. Compared with other protein fusions that have been used to study protein translocation, PSBT fusions have the advantage that they can be used to study the rate of the insertion process.     

Carboxymethyl-phenylalanine as a replacement for phosphotyrosine in SH2 domain binding

The crystal structure of human p56(lck) SH2 domain in complex with an inhibitor containing the singly charged p-(carboxymethyl)phenylalanine residue (cmF) as a phosphotyrosine (Tyr(P) or pY) replacement has been determined at 1.8 A resolution. The binding mode of the acetyl-cmF-Glu-Glu-Ile (cmFEEI) inhibitor is very similar to that of the pYEEI inhibitor, confirming that the cmFEEI inhibitor has a similar mechanism of SH2 domain inhibition despite its significantly reduced potency. Observed conformational differences in the side chain of the cmF residue can be interpreted in terms of maintaining similar interactions with the SH2 domain as the Tyr(P) residue. The crystal structure of the free p56(lck) SH2 domain has been determined at 1.9 A resolution and shows an open conformation for the BC loop and an open phosphotyrosine binding pocket, in contrast to earlier studies on the src SH2 domain that showed mostly closed conformation. The structural information presented here suggests that the carboxymethyl-phenylalanine residue may be a viable Tyr(P) replacement and represents an attractive starting point for the design and development of SH2 domain inhibitors with better pharmaceutical profiles.     

The antigen-binding domain of a human IgG-anti-F(ab')2 autoantibody

Recent studies revealed an immunoregulatory role of natural IgG-anti-F(ab')2 antibodies in both healthy individuals and patients with certain diseases. The implication of anti-F(ab')2 antibodies in the pathogenesis of diseases prompted us to study the gene segment structure of their antigen-binding domains and their binding characteristics. cDNA was prepared from the lymphocytes of a patient with a high IgG-anti-F(ab')2 serum titer. Variable heavy and light gene segments were amplified by PCR and inserted into a phagemid surface expression vector. Single-chain antibodies displayed on the phage surface were screened for binding to F(ab')2 fragments. The subsequent analysis of 95 single clones demonstrated that they all bound specifically to F(ab')2. Sequence analyses of 12 clones showed that 11 were identical and 1 contained a silent point mutation in the heavy chain and three amino acid exchanges in the light chain. The heavy chains belonged to the V(H)3 and the light chains to the V(kappa)2 gene family. The 11 identical light-chain genes were completely homologous to a germ-line sequence (DPK-15). Binding assays showed that the single-chain antibodies bind to F(ab')2, but not to Fab, Fc, or intact IgG. This binding pattern was confirmed by surface plasmon resonance studies, which revealed a relatively high affinity (Ka = 2.8 x 10(7) M(-1)). The strong binding capacity was further demonstrated by competitive inhibition of the serum anti-IgG antibody's interaction with antigen. The present study defines for the first time to our knowledge the gene segment structure of the antigen-binding domain of two human IgG-anti-F(ab')2 autoantibody clones and describes the binding kinetics of the purified monomeric fragments.     

The effect of different corticosteroids and cyclosporin A on interleukin-4 and interleukin-5 release from murine TH2-type T cells

By secretion of interleukin-4 and interleukin-5, TH2-type T cells are thought to play an important role in the pathogenesis of asthma. Corticosteroids are currently the most effective therapy available for asthma, but recently it has been demonstrated that cyclosporin A improves lung function in patients with severe corticosteroid-dependent asthma. In order to examine the effects of corticosteroids and cyclosporin A on anti-CD3-induced production of interleukin-4 and interleukin-5 we used the murine TH2-type cell clone D10.G4.1. Interleukin-4/interleukin-5 release was inhibited by all drugs tested with the following IC50 values (nmol/l) for interleukin-4 and interleukin-5, respectively: budesonide (0.32/0.22), beclomethasone (0.65/0.33), dexamethasone (4.70/3.52), 6 alpha-methyl-prednisolone (24.04/17.02), hydrocortisone (34.27/22.55), and cyclosporin A (72.59/242.21). In conclusion, corticosteroids exert strong inhibitory effects on cytokine production by TH2-cells, which may explain, at least partly, its clinical efficacy in asthma. Cyclosporin A also showed a concentration-dependent inhibition; however, in relation to corticosteroids the inhibitory activity of cyclosporin A was found to be weaker.     

The membrane topology of human transient receptor potential 3 as inferred from glycosylation-scanning mutagenesis and epitope immunocytochemistry

Transient receptor potential (Trp) proteins form ion channels implicated in the calcium entry observed after stimulation of the phospholipase C pathway. Kyte-Doolittle analysis of the amino acid sequence of Trp proteins identifies seven hydrophobic regions (H1-H7) with potential of forming transmembrane segments. A limited sequence similarity to voltage-gated calcium channel alpha1 subunits lead to the prediction of six transmembrane (TM) segments flanked by intracellular N and C termini and a putative pore region between TM5 and TM6. However, experimental evidence supporting this model is missing. Using human Trp 3 to test Trp topology, we now confirm the intracellular nature of the termini by immunocytochemistry. We also demonstrate presence of a unique glycosylation site in position 418, which defines one extracellular loop between H2 and H3. After removal of this site and insertion of ten separate glycosylation sites, we defined two additional extracellular loops between H4 and H5, and H6 and H7. This demonstrated the existence of six transmembrane segments formed of H2-H7. Thus, the first hydrophobic region of Trp rather than being a transmembrane segment is intracellular and available for protein-protein interactions. A site placed in the center of the putative pore region was glycosylated, suggesting that this region may have been luminal and was reinserted into the membrane at a late stage of channel assembly.     

The N-terminal part of the enzyme component (C2I) of the binary Clostridium botulinum C2 toxin interacts with the binding component C2II and functions as a carrier system for a Rho ADP-ribosylating C3-like fusion toxin

PURPOSE: The major obstacle to successful discordant kidney xenotransplantation is hyperacute rejection (HAR). Complement plays a key role in the induction of HRA, defined by endothelial cell activation, loss of vascular integrity, hemorrhage and thrombosis. The activation of complement is tightly controlled by a number of species-specific regulatory proteins which inhibit, at different points, the cascade of events leading to the formation of the membrane attack complex (MAC). We have tested the hypothesis that kidneys derived from transgenic mice expressing two human complement inhibitors, Decay Accelerating Factor (hDAF) and Membrane Cofactor Protein (MCP), could be protected from human complement-mediated damage. MATERIALS AND METHODS: Control and transgenic mice were perfused with human plasma by cannulation of the right jugular vein, at a perfusion rate of 10 microL./min. for two hours. Complement C3 deposition was detected on kidney sections by immunohistochemistry using specific FITC antibody. Complement-induced tissue damage was evaluated by histopathological examination. RESULTS: Heavy deposition of complement C3 was observed on kidneys derived from perfused control mice. This was associated with a characteristic HAR pathology of severe interstitial hemorrhage, inflammatory reaction, loss of glomerula and tubuli structure. Kidneys derived from mice transgenic for hDAF or hMCP were partially protected from both complement C3 deposition and tissue damage. The expression of both hDAF and hMCP in double transgenic mice significantly increases the protection from human complement-mediated damage. CONCLUSION: A novel model of in vivo perfusion with human plasma has been adopted to recreate the initial event of HAR. Our data show that this murine model could be very valuable to determine the effect of transgenic human molecules in protecting vascularized organs from human complement attack.     

Distinct regulation of glucose transport by interleukin-3 and oncogenes in a murine bone marrow-derived cell line

Growth factors and oncogenes promote glucose uptake, but the extent to which increased uptake is regulated at the level of glucose transporter function has not been clearly established. In this paper, we show that interleukin-3 (IL-3), a cytokine growth factor, and the transforming oncogenes ras and abl alter the activation state of glucose transporters by distinct mechanisms. Using bone marrow-derived IL-3-dependent 32Dc13 (32D clone 3) cells and 32D cells transformed with ras and abl oncogenes, we demonstrated that IL-3 enhanced [3H]-2-deoxyglucose (2-DOG) uptake in parental 32Dc13 cells by 40-50% at 0.2 mM 2-DOG, and this was associated with a 2.5-fold increase in transporter affinity for glucose (reduced Km). In comparison, ras and abl oncogenes enhanced 2-DOG uptake by 72-112%, associated with a 2-fold greater transporter affinity for glucose. The tyrosine kinase inhibitor genistein reversed the effects of both IL-3 and oncogenes on glucose uptake and reduced transporter affinity for glucose. Likewise, with exponentially growing 32D cells in the presence of IL-3, a protein kinase C inhibitor, staurosporine, and a phosphatidylinositol 3-kinase (PI-3) kinase inhibitor, wortmannin, inhibited 2-DOG uptake and decreased transporter affinity for glucose. In contrast, in oncogene-transformed cells, staurosporine inhibited 2-DOG uptake but failed to decrease transporter affinity for glucose, whereas wortmannin did not affect 2-DOG uptake. Inhibition of protein tyrosine phosphatases with vanadate enhanced 2-DOG uptake and transporter affinity for glucose in parental cells and in ras-transformed cells but had little effect on abl-transformed cells. Consistently, the serine/threonine phosphatase type 2A inhibitor okadaic acid enhanced 2-DOG uptake and transporter affinity for glucose in parental cells but had little effect on ras- or abl-transformed cells. These results demonstrate differences in the regulation of glucose transport in parental and oncogene-transformed 32D cells. Thus, IL-3 responses are dependent upon tyrosine, serine/threonine, and PI-3 kinases, whereas ras and abl effects on glucose transport depend upon tyrosine phosphorylation but are compromised in their dependence upon serine/threonine and PI-3 kinases.     

In vitro expression of the diphtheria toxin A-chain gene under the control of human chorionic gonadotropin gene promoters as a means of directing toxicity to ovarian cancer cell lines

OBJECTIVE: Our goal was to determine whether toxicity of the diphtheria toxin A-chain gene regulated by the human chorionic gonadotropin promoter can be directed to malignant ovarian cell lines. STUDY DESIGN: Plasmids containing diphtheria toxin A-chain gene linked to the regulatory elements of the metalloergothioneine and human chorionic gonadotropin promoters were transfected into the cell lines. Expression of diphtheria toxin A-chain gene was determined by the inhibition of a cotransfected luciferase reporter gene. RESULTS: Cytotoxicity of the diphtheria toxin A-chain gene is shown in a dose-responsive manner. Transfection of a plasmid expressing the diphtheria toxin A-chain gene controlled by a constitutive promoter readily inhibits protein synthesis. Specific inhibition of luciferase protein synthesis occurs in ovarian cancer cells transfected with the diphtheria toxin A-chain gene under the control of the human chorionic gonadotropin promoters when compared with normal ovarian epithelial cells or fibroblasts. CONCLUSIONS: These data demonstrate the preferential expression of the diphtheria toxin A-chain gene, regulated by the human chorionic gonadotropin promoter, to ovarian cancer cell lines. This provides an avenue for targeting such cells for suicide, toxin, or cytokine genes.     

Structure-function analysis of the Bcl-2 oncoprotein. Addition of a heterologous transmembrane domain to portions of the Bcl-2 beta protein restores function as a regulator of cell survival

The bcl-2 gene can potentially encode 26- and 22-kDa proteins that differ only in their carboxyl tails because of an alternative splicing mechanism. The larger of these proteins contains a hydrophobic transmembrane domain within its carboxyl terminus, resides (at least in part) in mitochondrial membranes and has been shown to prolong cell survival by blocking programmed cell death (also termed "apoptosis"). To explore the function of the shorter 22-kDa Bcl-2 protein that lacks a transmembrane domain, DNAs encoding p26-Bcl-2-alpha or p22-Bcl-2-beta were expressed in an interleukin-3 (IL-3)-dependent hematopoietic cell line 32D. In contrast to p26-Bcl-2 alpha that markedly prolonged cell survival, p22-Bcl-2-beta did not extend the survival of 32D cells when cultured in the absence of IL-3. Expression in 32D cells of a chimeric DNA that fused portions of the open reading frame common to Bcl-2-alpha and Bcl-2-beta (amino-acids 1-195) with sequences encoding the transmembrane and cytosolic domains of the IL-2 receptor-alpha protein resulted in production of a Bcl-2/IL-2R fusion protein that was capable of prolonging 32D cell survival in the setting of IL-3 withdrawal. Based on fractionation of cells to produce crude heavy membrane, light membrane, nuclei, and cytosolic preparations, much of the p22-Bcl-2-beta protein appeared to reside in the cytosol, whereas Bcl-2-alpha and the Bcl-2/IL-2R chimeric proteins were found exclusively in fractions that also contained the inner mitochondrial membrane protein F1-beta-ATPase. Taken together, these findings demonstrate the importance of membrane association for the function and intracellular targeting of the apoptosis-blocking Bcl-2 protein. Furthermore, despite the strong evolutionary conservation of the carboxyl regions of Bcl-2-alpha proteins observed previously for mammalian and avian species, these data suggest that a heterologous transmembrane domain can be substituted without loss of function.