Terminal Transmembrane Domain Research Articles

The carboxy terminal transmembrane domain of SPL7 mediates interaction with RAN1 at the endoplasmic reticulum to regulate ethylene signalling in Arabidopsis.

In Arabidopsis, copper (Cu) transport to the ethylene receptor ETR1 mediated using RAN1, a Cu transporter located at the endoplasmic reticulum (ER), and Cu homeostasis mediated using SPL7, the key Cu-responsive transcription factor, are two deeply conserved vital processes. However, whether and how the two processes interact to regulate plant development remain elusive. We found that its C-terminal transmembrane domain (TMD) anchors SPL7 to the ER, resulting in dual compartmentalisation of the transcription factor. Immunoprecipitation coupled mass spectrometry, yeast-two-hybrid assay, luciferase complementation imaging and subcellular co-localisation analyses indicate that SPL7 interacts with RAN1 at the ER via the TMD. Genetic analysis revealed that the ethylene-induced triple response was significantly compromised in the spl7 mutant, a phenotype rescuable by RAN1 overexpression but not by SPL7 without the TMD. The genetic interaction was corroborated by molecular analysis showing that SPL7 modulates RAN1 abundance in a TMD-dependent manner. Moreover, SPL7 is feedback regulated by ethylene signalling via EIN3, which binds the SPL7 promoter and represses its transcription. These results demonstrate that ER-anchored SPL7 constitutes a cellular mechanism to regulate RAN1 in ethylene signalling and lay the foundation for investigating how Cu homeostasis conditions ethylene sensitivity in the developmental context.

Proteomics Computational Analyses Suggest that the Antennavirus Glycoprotein Complex Includes a Class I Viral Fusion Protein (α-Penetrene) with an Internal Zinc-Binding Domain and a Stable Signal Peptide

A metatranscriptomic study of RNA viruses in cold-blooded vertebrates identified two related viruses from frogfish (Antennarius striatus) that represent a new genus Antennavirus in the family Arenaviridae (Order: Bunyavirales). Computational analyses were used to identify features common to class I viral fusion proteins (VFPs) in antennavirus glycoproteins, including an N-terminal fusion peptide, two extended alpha-helices, an intrahelical loop, and a carboxyl terminal transmembrane domain. Like mammarenavirus and hartmanivirus glycoproteins, the antennavirus glycoproteins have an intracellular zinc-binding domain and a long virion-associated stable signal peptide (SSP). The glycoproteins of reptarenaviruses are also class I VFPs, but do not contain zinc-binding domains nor do they encode SSPs. Divergent evolution from a common progenitor potentially explains similarities of antennavirus, mammarenavirus, and hartmanivirus glycoproteins, with an ancient recombination event resulting in a divergent reptarenavirus glycoprotein.

Investigating the interaction between peptides of the amphipathic helix of Hcf106 and the phospholipid bilayer by solid-state NMR spectroscopy

The chloroplast twin arginine translocation (cpTat) system transports highly folded precursor proteins into the thylakoid lumen using the protonmotive force as its only energy source. Hcf106, as one of the core components of the cpTat system, is part of the precursor receptor complex and functions in the initial precursor-binding step. Hcf106 is predicted to contain a single amino terminal transmembrane domain followed by a Pro-Gly hinge, a predicted amphipathic α-helix (APH), and a loosely structured carboxy terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes. To better understand the membrane active capabilities of Hcf106, we used solid-state NMR spectroscopy to investigate those properties of the APH. In this study, synthesized peptides of the predicted Hcf106 APH (amino acids 28–65) were incorporated at increasing mol.% into 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and POPC/MGDG (monogalactosyldiacylglycerol; mole ratio 85:15) multilamellar vesicles (MLVs) to probe the peptide–lipid interaction. Solid-state 31P NMR and 2H NMR spectroscopic experiments revealed that the peptide perturbs the headgroup and the acyl chain regions of phospholipids as indicated by changes in spectral lineshape, chemical shift anisotropy (CSA) line width, and 2H order SCD parameters. In addition, the comparison between POPC MLVs and POPC/MGDG MLVs indicated that the lipid bilayer composition affected peptide perturbation of the lipids, and such perturbation appeared to be more intense in a system more closely mimicking a thylakoid membrane.

Solid-state NMR investigations of peptide–lipid interactions of the transmembrane domain of a plant-derived protein, Hcf106

The chloroplast twin arginine translocation system transports highly folded precursor proteins across the thylakoid using the protonmotive force as its only energy source. Hcf106 and another thylakoid protein, cpTatC compose the precursor receptor complex. Hcf106 is predicted to contain a single amino terminal transmembrane domain (TMD) followed by a Pro–Gly hinge, an amphipathic α-helix, and a loosely structured carboxyl terminus. Hcf106 has been shown biochemically to insert spontaneously into thylakoid membranes; however, how this occurs is not understood. To investigate how Hcf106 inserts itself into the membrane unassisted, solid-state NMR spectroscopy was used to investigate the membrane activity of the TMD. A synthetic peptide of the Hcf106 TMD was incorporated into multilamellar vesicles made of 100% 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) or 85%:15% ratio with monogalactosyl diacylglycerol (POPC/MGDG) to probe peptide–lipid interaction. Solid-state 31P NMR and 2H NMR spectroscopic techniques were used to reveal peptide perturbations of the phospholipid membranes. Changes in spectral lineshape, chemical shift anisotropy width, 31P T1 relaxation time and SCD order parameters demonstrated that the Hcf106 TMD peptide interacted with the phospholipids. Furthermore, the comparison between POPC and POPC/MGDG multilamellar vesicles indicated that lipid bilayer composition affected the peptide–lipid interaction with the peptide interacting preferentially with vesicles that more closely mimic the thylakoid.

Molecular Architecture of OPA1, the Dynamin-Related GTPase Involved in Mitochondrial Fusion

Mitochondria are dynamic organelles that constantly undergo fusion and fission events. Four dynamin-related GTPases have been shown to facilitate mitochondrial dynamics. One of them, OPA1, mediates mitochondrial inner membrane fusion. Mutations in OPA1 have been linked to severe disease phenotypes characterized by progressive degeneration of the retinal ganglion cells, thus leading to blindness. OPA1 is a nuclear encoded protein that is inserted into the inner mitochondrial membrane through an N terminal transmembrane domain. There it undergoes proteolytic processing yielding a long, membrane-bound (OPA1) and a short, diffusible form (OPA1s). Fusion of the inner mitochondrial membrane requires both forms of OPA1, and the ability of OPA1 to bind and hydrolyze GTP. In previous structural studies, we have shown that recombinant OPA1s binds to lipid preparations with a composition similar to that of the inner mitochondrial membrane (Ban et al., 2010). Our findings indicated that the interaction of OPA1s with suitable membranes stimulates oligomerization and self-assembly. Using cryo-electron microscopy, we have visualized and analyzed the topological arrangement of OPA1 on these tubules and showed that the protein wraps around the perimeter of these tubes in a helical fashion. Interestingly, the protein formed a two-dimensional lattice on the lipid bilayer indicating that specific intermolecular interactions are at play. We are now investigating the molecular architecture of such OPA1-lipid assemblies using biochemical and high-resolution electron microscopy (EM) methods. Here, we report on strategies employed to generate substrates suitable for EM analysis and on our current insights into the properties and architecture of OPA1 assemblies. Reference: Ban, T., Heymann, J. A., Song, Z., Hinshaw, J. E., Chan, D. C.: OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation. Hum. Mol. Genet. 2010, 19: 2113-22.

Coarse Grain Simulations Reveal Movement of Synaptobrevin C Terminus in Response to Piconewton Forces Suggesting a Novel Fusion Pore Mechanism

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C terminal transmembrane (TM) helix. In coarse grain molecular dynamics simulations self-assembly of the membrane occurred with the syb2 TM helix inserted as expected from experimental data. The free energy profile for the position of the TM domain in the membrane was determined applying harmonic potentials to the peptide in its unbiased position, pulling it towards new biased equilibrium positions. The energy profile determined in this way predicts the energy landscapes for pulling syb2 towards the extravesicular side as expected for SNARE complex zippering. Applying a constant pulling force of 160 pN detaches the synaptobrevin C terminus from the vesicle's inner leaflet lipid head groups within ∼100ns and pulls the C terminus deeper into the membrane. This C terminal movement should occur on the physiological millisecond time scale at ∼120 pN force. It is facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. These results suggest a mechanism where fusion pore formation is induced by movement of the charged syb2 C terminus into the hydrophobic core of the membrane in response to the force generated by C terminal zippering of the SNARE complex. This displacement of the charged C terminus is expected to destabilize the membrane providing a plausible pathway to fusion pore formation. Supported by NIH grants R01GM085808, R21NS072577, EDICT Project grant 201924, an MRC fellowship and the Wellcome Trust.

Protein oligomerization mediated by the transmembrane carboxyl terminal domain of Bcl-XL

Bcl-XL is a pro-survival member of the Bcl-2 family that can be found in the outer mitochondrial membrane and in soluble cytosolic homodimers. Bcl-XL can bind pro-apoptotic members of this family preventing them from activating the execution phase of apoptosis. Bcl-XL has been shown to homodimerize in different ways, although most binding and structural assays have been carried out in the absence of its carboxyl terminal transmembrane domain. We show here that this domain can by itself direct protein oligomerization, which could be related to its previously reported role in mitochondrial morphology alterations and apoptosis inhibition. Structured summary of protein interactionsVamp2physically interactswithVamp2byblue native page(View interaction)Vamp2physically interactswithVamp2bycross-linking study(View interaction)Bcl-Xlphysically interactswithBcl-Xlbyblue native page(View interaction)Bcl-Xlphysically interactswithBcl-Xlbycross-linking study(View interaction)

Characterization of a novel Taenia solium oncosphere antigen

Infections due to Taenia solium in humans (taeniasis/cysticercosis) remain a complex health problem, particularly in developing countries. We identified two oncosphere proteins that might protect the porcine intermediate host against cysticercosis and therefore help prevent disease in humans. One of these proteins was further identified by two-dimensional gel electrophoresis and micro-sequencing. The gene encoding this protective protein was also identified, cloned and characterized. The native 31.5kDa protein Tso31 has four variants at the cDNA level. The longest sequence from which the others seem to derive, encodes a 253 amino acid peptide. The predicted protein has a molecular weight of 25.1kDa, one putative N-glycosylation site, two fibronectin type III domains, and one C terminal transmembrane domain. The gene structure of the protein consists of four exons and three introns. The finding of one gene and four different cDNAs for Tso31 suggests the existence of a possible mechanism of differential splicing in this parasite. The Tso31 protein is exclusive to T. solium oncospheres with a putative protein structure of an extra-cellular receptor-like protein. The Tso31 protein was expressed as a recombinant protein fused to GST and tested in a vaccine to determine its effectiveness in protecting pigs against cysticercosis. Only two pigs out of eight vaccinated were protected and although the total median number of cyst decreased in vaccinated pigs compared to controls this decrease was not statistically significant (P=0.09).

The NH2‐Terminal Transmembrane and Lumenal Domains of LGP85 are Needed for the Formation of Enlarged Endosomes/Lysosomes

LGP85 is a lysosomal membrane protein possessing a type III topology and is also known as a member of the CD36 superfamily of proteins, such as CD36 and the scavenger-receptor BI (SR-BI). We have recently demonstrated that overexpression of LGP85 in various mammalian cell lines causes the enlargement of endosomal/lysosomal compartments (ELCs). Using chimeras and deletion mutants, we show here that the lumenal region of LGP85 is necessary, but not sufficient, for the development of ELCs. Effective formation of enlarged ELC was largely dependent on the presence of a preceding NH(2)-terminal transmembrane segment. Analyses of deletion mutants within the lumenal domain further revealed a requirement of the NH(2)-terminal transmembrane proximal lumenal region, with high sequence similarity with SR-BI for the enlargement of ELC. These results suggest that an interaction of the NH(2)-terminal transmembrane proximal lumenal domain of LGP85 with the inner leaflet of endosomal/lysosomal membranes through the connection with the transmembrane domain is an essential determinant for the regulation of endosomal/lysosomal membrane traffic. Interestingly, although the NH(2)-terminal transmembrane domain itself was not sufficient for the enlargement of ELCs, it appeared to be required for direct targeting of LGP85 from the trans-Golgi network to late endosomes/lysosomes. Taken together, these results indicate the involvement of distinct domain of LGP85 in the targeting to, and biogenesis and maintenance of, ELC.

BNips: a group of pro-apoptotic proteins in the Bcl-2 family.

BNip (formerly known as Nip) proteins, including homologues isolated from human, mouse and Caenorhabditis. elegans, are a relatively new subgroup of the Bcl-2 family. These proteins are classified into this family based on limited sequence homology with the Bcl-2 homology domain 3 and carboxyl terminal transmembrane domain. BNip proteins were first discovered based on their interaction with the adenovirus E1B 19 kDa/Bcl-2 family protein and since then, their roles in cell death pathways have been actively studied. However, the precise mechanisms by which the BNip proteins induce apoptosis and/or necrosis remain to be determined. To advance our knowledge, we have provided a summary and review of current literature regarding BNip proteins including comparative sequence analysis, mutational mapping of the functional domains, and cell death mechanisms involving disruption of mitochondrial homeostasis. Since BNip proteins are expressed at high levels in the heart as compared to other organs, their roles in cardiomyocyte injury during hypoxia or viral infection is a focus of this review. Finally, we discuss potential directions for further study on this increasingly important group of pro-apoptotic proteins.

Conformational Changes That Occur during M3Muscarinic Acetylcholine Receptor Activation Probed by the Use of an in Situ Disulfide Cross-linking Strategy

The structural changes involved in ligand-dependent activation of G protein-coupled receptors are not well understood at present. To address this issue, we developed an in situ disulfide cross-linking strategy using the rat M(3) muscarinic receptor, a prototypical G(q)-coupled receptor, as a model system. It is known that a tyrosine residue (Tyr(254)) located at the C terminus of transmembrane domain (TM) V and several primarily hydrophobic amino acids present within the cytoplasmic portion of TM VI play key roles in determining the G protein coupling selectivity of the M(3) receptor subtype. To examine whether M3 receptor activation involves changes in the relative orientations of these functionally critical residues, pairs of cysteine residues were substituted into a modified version of the M(3) receptor that contained a factor Xa cleavage site within the third intracellular loop and lacked most endogenous cysteine residues. All analyzed mutant receptors contained a Y254C point mutation and a second cysteine substitution within the segment Lys(484)-Ser(493) at the intracellular end of TM VI. Following their transient expression in COS-7 cells, mutant receptors present in their native membrane environment (in situ) were subjected to mild oxidizing conditions, either in the absence or in the presence of the muscarinic agonist, carbachol. The successful formation of disulfide cross-links was monitored by studying changes in the electrophoretic mobility of oxidized, factor Xa-treated receptors on SDS gels. The observed cross-linking patterns indicated that M(3) receptor activation leads to structural changes that allow the cytoplasmic ends of TM V and TM VI to move closer to each other and that also appear to involve a major change in secondary structure at the cytoplasmic end of TM VI. This is the first study employing an in situ disulfide cross-linking strategy to examine agonist-dependent dynamic structural changes in a G protein-coupled receptor.

Molecular Basis for the Transport of Cytochrome P450 2E1 to the Plasma Membrane

Endoplasmic reticulum-resident cytochrome P450 enzymes that face the cytosol are present on the plasma membrane of hepatocytes, but the molecular origin for their transport to this compartment has until now remained unknown. The molecular basis for the transport of rat ethanol-inducible cytochrome P450 2E1 (CYP2E1) to the plasma membrane was investigated by transfection of several different mutant cDNAs into mouse H2.35 hepatoma cells. Two NH(2)-terminal CYP2E1 mutants were constructed: N(++)2E1, which carried two positive charges in the NH(2) terminus, and 2C-2E1, in which the transmembrane domain of CYP2E1 was replaced with that of CYP2C1, which was previously described to cause retention of CYP2C1 in the endoplasmic reticulum, as well as CYP2E1 COOH-terminally tagged with the vesicular stomatitis virus G protein (VSV-G) epitope (2E1-VSV-G). Immunofluorescent microscopy and cell surface biotinylation experiments revealed that all CYP2E1 variants were present on the extracellular side of the plasma membrane. The VSV-G epitope on CYP2E1 was detected on the outside of the plasma membrane using VSV-G-specific antibodies, indicating that the large COOH-terminal part of CYP2E1 is indeed exposed on the outside of the plasma membrane. The relative levels of CYP2E1, 2C-2E1, and 2E1-VSV-G on the cell surface were found to be about 2% of total cellular enzyme, whereas twice this amount of N(++)2E1 was recovered at the cell surface. Protease protection experiments performed on microsomes isolated from cDNA transfected cells revealed that a small fraction of CYP2E1 and all variant proteins was found to be located in the lumen of the endoplasmic reticulum (type II orientation), whereas the majority of the proteins were in the expected cytosolic or type I orientation. It is concluded that the NH(2)-terminal transmembrane domain of CYP2E1 plays a critical role in directing the protein to the cell surface and that topological inversion of a small fraction of CYP2E1 in the endoplasmic reticulum directs the protein to the plasma membrane.

Molecular cloning, chromosomal mapping and tissue-specific expression of a novel human alpha1,2-mannosidase gene involved in N-glycan maturation.

Class I alpha1,2-mannosidases play an essential role in the elaboration of complex and hybrid N -glycans in mammalian cells. Using degenerate primers based on amino acid sequences conserved in all members of this enzyme family for RT-PCR, two distinct PCR products were obtained from placenta and lymphocyte cDNAs. One of these was related to the previously cloned human and murine alpha1, 2-mannosidase IA whereas the other was very similar to murine alpha1, 2-mannosidase IB. Northern blot analysis of human tissues with these two alpha1,2-mannosidase probes revealed very different patterns of tissue-specific expression. Similar tissue-specific expression of alpha1,2-mannosidase IA and IB was also observed on Northern blots of adult mouse tissues. A human placenta cDNA library was screened and PCR of brain, placenta, and lymphocyte cDNAs was performed in order to isolate the human alpha1,2-mannosidase IB cDNA. This cDNA encodes a type II membrane protein of 73 kDa that is 94% identical in amino acid sequence to the murine alpha1,2-mannosidase IB (Herscovics et al., 1994, J. Biol. Chem., 269, 9864-9871). A truncated soluble form of the human alpha1,2-mannosidase IB lacking its N -terminal transmembrane domain was expressed as a secreted protein in Pichia pastoris . The recombinant enzyme was incubated with [3H]Man9GlcNAc and [3H]Man8GlcNAc (isomer B), and high performance liquid chromatography analysis of the products showed that [3H]Man9GlcNAc was readily converted to [3H]Man6GlcNAc and much more slowly to [3H]Man5GlcNAc, whereas [3H]Man8GlcNAc was rapidly trimmed to [3H]Man5GlcNAc. The human alpha1,2-mannosidase IB gene was isolated from a P1 human genomic library and shown to be at least 60 kb in size and to contain at least 13 exons. The gene was localized by fluorescence in situ hybridization to human chromosome 1p13, a region that undergoes many aberrations in various types of human cancers. These results show that there are at least two Class I alpha1,2-mannosidases in the human and murine genomes with very distinct transcriptional regulation in different tissues.

Syntaxin 11: A Member of the Syntaxin Family without a Carboxyl Terminal Transmembrane Domain

We have cloned a novel syntaxin-like molecule, designated human syntaxin 11 (hsyn11). The open reading frame encodes a polypeptide of 287 amino acids with potential coiled-coil domains. hsyn11 has extensive homology to members of the syntaxin family, particularly syntaxin 1 and syntaxin 2. Unlike other members of the syntaxin family, however, hsyn11 has a short cysteine-rich carboxyl-terminal tail but not a typical hydrophobic domain which may serve as a membrane anchor. Northern blot analysis revealed two transcripts of ∼0.8 kb and ∼1.7 kb in length that are particularly abundant in heart and placenta, although lower levels were also detectable in other tissues except in the brain. Consistent with the lack of a distinct membrane anchorage sequence in hsyn11, indirect immunofluorescence microscopy of transiently expressed N-terminally myc-tagged hsyn11 revealed a diffuse, cytoplasmic labeling.

Functional calcium release channel formed by the carboxyl-terminal portion of ryanodine receptor

The ryanodine receptor (RyR) is one of the key proteins involved in excitation-contraction (E-C) coupling in skeletal muscle, where it functions as a Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. RyR consists of a single polypeptide of approximately 560 kDa normally arranged in a homotetrameric structure, which contains a carboxyl (C)-terminal transmembrane domain and a large amino (N)-terminal cytoplasmic domain. To test whether the carboxyl-terminal portion of RyR is sufficient to form a Ca2+ release channel, we expressed the full-length (RyR-wt) and C-terminal (RyR-C, approximately 130 kDa) RyR proteins in a Chinese hamster ovary (CHO) cell line, and measured their Ca2+ release channel functions in planar lipid bilayer membranes. The single-channel properties of RyR-wt were found to be similar to those of RyR from skeletal muscle SR. The RyR-C protein forms a cation-selective channel that shares some of the channel properties with RyR-wt, including activation by cytoplasmic Ca2+ and regulation by ryanodine. Unlike RyR-wt, which exhibits a linear current-voltage relationship and inactivates at millimolar Ca2+, the channels formed by RyR-C display significant inward rectification and fail to close at high cytoplasmic Ca2+. Our results show that the C-terminal portion of RyR contains structures sufficient to form a functional Ca2+ release channel, but the N-terminal portion of RyR also affects the ion-conduction and calcium-dependent regulation of the Ca2+ release channel.

Coordinate expression of the three zona pellucida genes during mouse oogenesis

The mammalian zona pellucida is an extracellular matrix that surrounds growing oocytes, ovulated eggs and early embryos. The mouse zona is composed of three sulfated glycoproteins: ZP1, ZP2 and ZP3. Each is critically involved in fertilization, the postfertilization block to polyspermy and protection of the preimplantation embryo. We have previously isolated cDNAs encoding mouse ZP2 and ZP3 and now report the isolation of a full-length cDNA encoding ZP1. Mouse ZP1 is composed of a 623 amino acid polypeptide chain with a signal peptide and a carboxyl terminal transmembrane domain, typical of all zona proteins. Sequence comparison demonstrate that mouse ZP1 is an orthologue of a rabbit zona protein, R55. The expression of R55 has been reported previously in both oocytes and granulosa cells. However, by northern analysis and in situ hybridization with 33P-labelled antisense probes to each of the three mouse zona mRNAs, we have determined that the expression of each mouse zona gene is restricted to the oocyte. ZP2 transcripts, but not ZP1 or ZP3, are detected in resting (15 microns diameter) oocytes, and all three zona transcripts coordinately accumulate as oocytes begin to grow. Together they represent approximately 1.5% of the total poly(A)+ RNA in 50-60 microns oocytes. In the latter stages of oogenesis, their abundance declines and each zona transcript is present in ovulated eggs at less than 5% of its maximal level. No zona transcripts were detected above background signal in granulosa cells. We conclude that, in mice, the three zona pellucida genes are expressed in a coordinate, oocyte-specific manner during the growth phase of oogenesis. Our data support the hypothesis that the transcription of the zona genes is controlled, in part, by shared regulatory element(s).

Cloning and molecular characterization of mouse bcl-x in B and T lymphocytes.

We report the cloning and initial characterization of the mouse homologues of the bcl-2-related gene, bcl-x. We compare these to the two major isoforms of human bcl-x that were previously identified, bcl-xLong (bcl-xL) which has death repressor activity similar to bcl-2, and bcl-xShort (bcl-xS), an alternative mRNA splice product that deletes a highly conserved domain shared by bcl-2 family members and promotes cell death in transfection studies. In addition to murine (m) bcl-xL and mbcl-xS, we have cloned a novel cDNA isoform that we designate mbcl-x delta TM. This cDNA deletes, by means of alternative splicing, the carboxy terminal transmembrane domain of bcl-x and is predicted to be a soluble, rather than membrane-bound, protein. We found that mbcl-x mRNA is highly inducible in splenocytes stimulated with either anti-CD3 Abs or LPS/dextran sulfate, and that the major species of mbcl-x mRNA in a variety of cell lines and tissues was the xL isoform. Transfection of mbcl-xL or mbcl-xS into Hela cells resulted in targeting primarily to mitochondria, whereas mbcl-x delta TM localized diffusely throughout the cytosol. Overexpression of the novel delta TM isoform in an IL-3-dependent cell line delayed the onset of apoptosis induced by growth factor withdrawal. Thus, a naturally-occurring form of mbcl-x present in lymphocytes that does not localize to the mitochondrial membrane is functional in preventing apoptotic cell death. Moreover, these data suggest that bcl-x provides a life signal in activated lymphocytes.