Sequence-specific dimerization of the transmembrane domain of the "BH3-only" protein BNIP3 in membranes and detergent

Mitochondria-mediated apoptosis is regulated by proteins of the Bcl-2 superfamily, most of which contain a C-terminal hydrophobic domain that plays a role in membrane targeting. Experiments with BNIP3 have implicated the transmembrane (TM) domain in its proapoptotic function, homodimerization, and interactions with Bcl-2 and Bcl-xL. We show that the BNIP3 TM domain self-associates strongly in Escherichia coli cell membranes and causes reversible dimerization of a soluble protein in the detergent SDS when expressed as an in-frame fusion. Limited mutational analysis identifies specific residues that are critical for BNIP3 TM self-association in membranes, and these residues are also important for dimerization in SDS micelles, suggesting that the self-association observed in membranes is preserved in detergent. The effects of sequence changes at positions Ala176 and Gly180 suggest that the BNIP3 TM domain associates using a variant of the GXXXG motif previously shown to be important in the dimerization of glycophorin A. The importance of residue His173 in BNIP3 TM domain dimerization indicates that polar residues, which have been implicated in self-association of model TM peptides, can act in concert with the AXXXG motif to stabilize TM domain interactions. Our results demonstrate that the hydrophobic C-terminal TM domain of the pro-apoptotic BNIP3 protein dimerizes tightly in lipidic environments, and that this association has a strong sequence dependence but is independent of the identity of flanking regions. Thus, the transmembrane domain represents another region of the Bcl-2 superfamily of proteins that is capable of mediating strong and specific protein-protein interactions.     

The retinal rod Na(+)/Ca(2+),K(+) exchanger contains a noncleaved signal sequence required for translocation of the N terminus

The retinal rod Na(+)/Ca(2+),K(+) exchanger (RodX) is a polytopic membrane protein found in photoreceptor outer segments where it is the principal extruder of Ca(2+) ions during light adaptation. We have examined the role of the N-terminal 65 amino acids in targeting, translocation, and integration of the RodX using an in vitro translation/translocation system. cDNAs encoding human RodX and bovine RodX through the first transmembrane domain were correctly targeted and integrated into microsomal membranes; deletion of the N-terminal 65 amino acids (aa) resulted in a translation product that was not targeted or integrated. Deletion of the first 65 aa had no effect on membrane targeting of full-length RodX, but the N-terminal hydrophilic domain no longer translocated. Chimeric constructs encoding the first 65 aa of bovine RodX fused to globin were translocated across microsomal membranes, demonstrating that the sequence could function heterologously. Studies of fresh bovine retinal extracts demonstrated that the first 65 aa are present in the native protein. These data demonstrate that the first 65 aa of RodX constitute an uncleaved signal sequence required for the efficient membrane targeting and proper membrane integration of RodX.     

Signal recognition protein is required for the integration of acetylcholine receptor delta subunit, a transmembrane glycoprotein, into the endoplasmic reticulum membrane

Purified Signal Recognition Protein (SRP) has previously been shown to be required for the translocation of secretory proteins across the microsomal membrane (Walter and Blobel, 1980. Proc. Natl. Acad. Sci. U. S. A. 77:7, 112-7, 116) and to function in the early events of this process (Walter and Blobel, 1981. J. Cell Biol. 91:557-561). We demonstrate here that the delta subunit of acetylcholine receptor (AChR- delta), a transmembrane glycoprotein, likewise requires SRP for its asymmetric integration into microsomal membranes. We further demonstrate by partial sequence analysis that AChR-delta is synthesized with a transient NH2-terminal signal sequence of 21 residues that is cleaved off during integration into microsomal membranes. Integration of AChR-delta into the microsomal membrane vesicles proceeded asymmetrically, yielding a large (44 kdalton) core-glycosylated domain, inaccessible to externally added proteolytic enzymes and a smaller (approximately 16 kdalton) domain exposed on the outside of the vesicles and accessible to externally added proteolytic enzymes. The NH2 terminus of the molecule is contained in the 44-kdalton domain.     

Mutations in the NH2-terminal domain of the signal peptide of preproparathyroid hormone inhibit translocation without affecting interaction with signal recognition particle

The amino-terminal domain of a eukaryotic signal peptide, from bovine parathyroid hormone, was altered by in vitro mutagenesis of the cDNA. The function of "internalized" signal sequence mutants and of deletion mutants was assayed using an in vitro translation-translocation system. The addition of 11 amino acids to the NH2 terminus of the signal peptide did not prevent normal processing of the precursor protein, whereas a 23-amino acid extension blocked processing. These data suggest that the NH2-terminal sequences of internal signal peptides must be permissive of the signal function. Deletion of 6 NH2-terminal amino acids from the signal peptide had no effect on its cleavage by microsomal membranes, but removal of 10 or 13 amino acids, including all charged residues prior to the hydrophobic core, prevented processing. For both the extension and deletion mutations, processed proteins were protected from proteolytic digestion, whereas unprocessed forms were not, which indicated that the unprocessed mutant proteins were not translocated across the microsomal membrane. Translation of both the extension and deletion translocation-deficient mutants was arrested by signal recognition particle, and salt-washed microsomal membranes reversed the translational arrest. These data demonstrate that the NH2-terminal domain is not required for the interaction of signal recognition particle with the signal peptide or with signal recognition particle receptor, but is required for formation of a maximally translocation-competent complex with the microsomal membrane.     

A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function.

The biosynthesis of a secretory protein and a transmembrane viral glycoprotein are compared by two different experimental approaches. (a) NH2-terminal sequence analysis has been performed on various forms of the transmembrane glycoprotein of vesicular stomatitis virus synthesized in cell-free systems. The sequence data presented demonstrate that the nascent precursor of the glycoprotein contains a "signal sequence" of 16 amino acids at the NH2 terminus, whose sequence is Met-Lys-Cys-Leu-Leu-Tyr-Leu-Ala-Phe-Leu-Phe-Ile-(His-Val-Asn)-Cys. This signal sequence is proteolytically cleaved during the process of insertion into microsomal membranes prior to chain completion. The new NH2 terminus of the inserted, cleaved, and glycosylated membrane protein is located within the lumen of the microsomal vesicles and is identical to that of the authentic glycoprotein from virions. (b) Nascent chain competition experiments were performed between this glycoprotein, bovine pituitary prolactin (a secretory protein), and rabbit globin (a cytosolic protein). It was found that the nascent membrane glycoprotein, but not nascent globin, competed with nascent prolactin for membrane sites involved in the early biosynthetic event of transfer across membranes. These data suggest that an initially common pathway is involved in the biogenesis of secretory proteins and at least one class of integral membrane proteins.     

Characterization of membrane association domains within the Tomato ringspot nepovirus X2 protein, an endoplasmic reticulum-targeted polytopic membrane protein

Replication of nepoviruses (family Comoviridae) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleoside triphosphate-binding protein (NTB) of Tomato ringspot nepovirus is an integral membrane protein with two ER-targeting sequences and have suggested that it anchors the viral replication complex (VRC) to the membranes. A second highly hydrophobic protein domain (X2) is located immediately upstream of the NTB domain in the RNA1-encoded polyprotein. X2 shares conserved sequence motifs with the comovirus 32-kDa protein, an ER-targeted protein implicated in VRC assembly. In this study, we examined the ability of X2 to associate with intracellular membranes. The X2 protein was fused to the green fluorescent protein and expressed in Nicotiana benthamiana by agroinfiltration. Confocal microscopy and membrane flotation experiments suggested that X2 is targeted to ER membranes. Mutagenesis studies revealed that X2 contains multiple ER-targeting domains, including two C-terminal transmembrane helices and a less-well-defined domain further upstream. To investigate the topology of the protein in the membrane, in vitro glycosylation assays were conducted using X2 derivatives that contained N-glycosylation sites introduced at the N or C termini of the protein. The results led us to propose a topological model for X2 in which the protein traverses the membrane three times, with the N terminus oriented in the lumen and the C terminus exposed to the cytoplasmic face. Taken together, our results indicate that X2 is an ER-targeted polytopic membrane protein and raises the possibility that it acts as a second membrane anchor for the VRC.     

Study of the secondary structure of the C-terminal domain of the antiapoptotic protein bcl-2 and its interaction with model membranes

Bcl-2 is a protein which inhibits programmed cell death. It is associated to many cell membranes such as mitochondrial outer membrane, endoplasmic reticulum, and nuclear envelope, apparently through a C-terminal hydrophobic domain. We have used infrared spectroscopy to study the secondary structure of a synthetic peptide (a 23mer) with the same sequence as this C-terminal domain (residues 217-239) of Bcl-2. The spectrum of this peptide in D(2)O buffer shows an amide I' band with a maximum at 1622 cm(-1), which clearly indicates its tendency to aggregate in aqueous solvent. However, the peptide incorporated in multilamellar phosphatidylcholine membranes shows a totally different spectrum of the amide I' band, with a maximum at 1655 cm(-)(1), indicating a predominantly alpha-helical structure. Addition of the peptide to unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein. Differential scanning calorimetry of dimyristoylphosphatidylcholine multilamellar vesicles in which the peptide was incorporated revealed that increasing concentrations of the peptide progressively broadened the pretransition and the main transition, as is to be expected for a membrane integral molecule. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in fluid phosphatidylcholine vesicles showed that increasing concentrations of the peptide produced increased polarization values, pointing to an increase in the apparent order of the membrane and indicating that high concentrations of the peptide considerably broaden the phase transition of dimyristoylphosphatidylcholine multilamellar vesicles. Quenching the intrinsic fluorescence of the Tyr-235 of the peptide, by KI, indicated that this aminoacyl residue is highly exposed to aqueous solvent when incorporated in phospholipid vesicles. The results are discussed in terms of their relevance to the proposed topology of insertion of Bcl-2 into biological membranes.     

Role of signal recognition particle in the membrane assembly of Sindbis viral glycoproteins

We have investigated the role of signal recognition particle (SRP) in the biosynthesis of Sindbis glycoproteins by translating the viral 26S mRNA in a wheat-germ cell-free system. SRP was shown to have no effect on the synthesis or proteolytic processing of the cytoplasmic C protein. In contrast, the membrane integration and the proteolytic processing of the viral glycoproteins PE2 and E1 were demonstrated to be SRP-dependent. In the absence of microsomal membranes, SRP caused an arrest of the synthesis of the viral glycoproteins. This arrest could be released by the addition of salt-extracted microsomal membranes. Synchronization experiments indicated that the uncleaved signal sequence of PE2 was recognized by SRP after at most 130 amino acids of PE2 had been polymerized. No apparent interaction of SRP with a putative signal sequence of E1 and/or a 6-kDa peptide could be detected.     

Characterization of the signal that directs Bcl-x(L), but not Bcl-2, to the mitochondrial outer membrane

It is assumed that the survival factors Bcl-2 and Bcl-x(L) are mainly functional on mitochondria and therefore must contain mitochondrial targeting sequences. Here we show, however, that only Bcl-x(L) is specifically targeted to the mitochondrial outer membrane (MOM) whereas Bcl-2 distributes on several intracellular membranes. Mitochondrial targeting of Bcl-x(L) requires the COOH-terminal transmembrane (TM) domain flanked at both ends by at least two basic amino acids. This sequence is a bona fide targeting signal for the MOM as it confers specific mitochondrial localization to soluble EGFP. The signal is present in numerous proteins known to be directed to the MOM. Bcl-2 lacks the signal and therefore localizes to several intracellular membranes. The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM. These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-x(L) specifically functions on the MOM.     

Functional expression of bovine 17 alpha-hydroxylase in COS 1 cells is dependent upon the presence of an amino-terminal signal anchor sequence.

Microsomal forms of eukaryotic cytochrome P450 proteins are integral membrane proteins of the endoplasmic reticulum (ER) membrane which are targeted to the ER via the signal recognition particle pathway. A hydrophobic amino terminus serves as a combined signal sequence and major membrane anchor (signal-anchor sequence) for the microsomal P450s. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the ER of COS 1 cells in order to evaluate the role of membrane insertion of the amino-terminal signal-anchor of microsomal P450s as a functional determinant for these enzymes. Previously, we have shown that deletion of the hydrophobic amino terminus from P45017 alpha reduced membrane targeting and insertion by 5-fold compared with the wild-type protein, abolished enzymatic activity, and resulted in an aberrant CO difference spectrum. In the present study we have replaced the amino terminus of P45017 alpha with two heterologous signal-anchor sequences, one that is similar and one that is very different from the P45017 alpha sequence. The chimeric proteins were expressed in COS 1 cells. Immunoblot analysis of isolated microsomal membranes show that the heterologous signal-anchor sequences functioned to target the P45017 alpha protein to the ER. Enzymatic assays in intact COS 1 cells indicate that both the chimeric proteins are efficient 17 alpha-hydroxylase enzymes. The amino terminus of P45017 alpha was also replaced with a sequence that is not a signal-anchor, and the expressed protein was neither targeted to the ER nor was functional in COS 1 cells. In conclusion, both the structure and catalytic activity of P45017 alpha in COS 1 cells is dependent upon an amino-terminal sequence that functions as a signal-anchor sequence and not upon the precise sequence of the amino terminus.     

Accessibility of microsomal membrane proteins for protease K]

The effect of protease K on the microsomal membrane proteins was studied. It was shown that treatment of the microsomal membranes by low concentrations of protease did not remove more than 35% of total membrane proteins. Treatment by higher protease concentrations removed about 50% of the proteins. The residual protein is accessible to protease after treatment of the membranes by phospholipase A2. A simultaneous treatment of the microsomal membrane by protease and phospholipase removed up to 80% of the membrane proteins. In this way about 30% of microsomal membrane proteins are protected by the phospholipid against the action of protease K.     

Characterization of an anti-apoptotic glycoprotein encoded by Kaposi's sarcoma-associated herpesvirus which resembles a spliced variant of human survivin

We have investigated the expression and function of a novel protein encoded by open reading frame (ORF) K7 of Kaposi's sarcoma-associated herpesvirus (KSHV). Computational analyses revealed that K7 is structurally related to survivin-DeltaEx3, a splice variant of human survivin that protects cells from apoptosis by an undefined mechanism. Both K7 and survivin-DeltaEx3 contain a mitochondrial-targeting sequence, an N-terminal region of a BIR (baculovirus IAP repeat) domain and a putative BH2 (Bcl-2 homology)-like domain. These suggested that K7 is a new viral anti-apoptotic protein and survivin-DeltaEx3 is its likely cellular homologue. We show that K7 is a glycoprotein, which can inhibit apoptosis and anchor to intracellular membranes where Bcl-2 resides. K7 does not associate with Bax, but does bind to Bcl-2 via its putative BH2 domain. In addition, K7 binds to active caspase-3 via its BIR domain and thus inhibits the activity of caspase-3. The BH2 domain of K7 is crucial for the inhibition of caspase-3 activity and is therefore essential for its anti-apoptotic function. Furthermore, K7 bridges Bcl-2 and activated caspase-3 into a protein complex. K7 therefore appears to be an adaptor protein and part of an anti-apoptotic complex that presents effector caspases to Bcl-2, enabling Bcl-2 to inhibit caspase activity. These data also suggest that survivin-DeltaEx3 might function by a similar mechanism to that of K7. We denote K7 as vIAP (viral inhibitor-of-apoptosis protein).     

Beta-galactosidase fused to the hydrophobic domain of cytochrome b5 spontaneously associates with liposomes

Since liver microsomal cytochrome b5 spontaneously associates with liposomes and membranes by means of its C-terminal hydrophobic domain (HP), chimeric proteins containing HP prepared by genetic fusion might also spontaneously associate with liposomes or cellular membranes. Synthetic DNA corresponding to the hydrophobic domain of cytochrome b5 was enzymatically fused in-frame to cloned DNA corresponding to the C-terminus of the Escherichia coli enzyme, beta-galactosidase. This protein, LacZ:HP, synthesized in E. coli and purified from a crude E. coli membrane extract, was shown to spontaneously associated with liposomes, as does cytochrome b5. Association is rapid and stable in the presence of salt and high pH and the fusion protein behaves as an integral membrane protein. LacZ:HP can be readily and extensively purified from crude extracts by association with liposomes and this procedure may provide a convenient purification scheme for proteins not otherwise readily purified, for example polypeptides from cloned gene fragments to be used for antibody production. These hybrid proteins may represent a new potentially useful class of polypeptides capable of hydrophobic interactions with membranes.     

Topogenic analysis of the human immunodeficiency virus type 1 envelope glycoprotein, gp160, in microsomal membranes

The orientation in cellular membranes of the 856 amino acid envelope glycoprotein precursor, gp160, of human immunodeficiency virus type 1 was investigated in vitro. Variants of the env gene were transcribed using the bacteriophage SP6 promoter, translated using a rabbit reticulocyte lysate, and translocated into canine pancreatic microsomal membranes. Immunoprecipitation studies of gp160 variants using antibodies specific for various gp160-derived polypeptides provided evidence that the external (cell surface) domain of gp160 begins at the mature amino terminus of the protein and continues through amino acid 665. A stop-transfer sequence (transmembrane domain) was identified in a hydrophobic region COOH-terminal to amino acid 665 and NH2-terminal to amino acid 732. Protease protection experiments demonstrated that gp160 possesses a single cytoplasmic domain COOH-terminal to residue 707. Membrane extraction studies using carbonate buffer provided evidence that the 29 amino acid hydrophobic domain (residues 512-541) of gp160 was unable to serve as a stop-transfer sequence. Finally, we propose that the cytoplasmic tail of gp160 forms a secondary association with the microsomal membranes.     

New insights in the role of Bcl-2 Bcl-2 and the endoplasmic reticulum

The oncogenic protein Bcl-2 which is expressed in membranes of different subcellular organelles protects cells from apoptosis induced by endogenic stimuli. Most of the results published so far emphasise the importance of Bcl-2 at the mitochondria. Several recent observations suggest a role of Bcl-2 at the endoplasmic reticulum (ER). Bcl-2 located at the ER was shown to interfere with apoptosis induction by Bax, ceramides, ionising radiation, serum withdrawal and c-myc expression. Although the detailed functions of Bcl-2 at the ER remain elusive, several speculative mechanisms may be supposed. For instance, Bcl-2 at the ER may regulate calcium fluxes between the ER and the mitochondria. In addition, Bcl-2 is able to interact with the endoplasmic protein Bap31 thus avoiding caspase activation at the ER. Bcl-2 may also abrogate the function of ER located pro-apoptotic Bcl-2 like proteins by heterodimerization. Current data on the function of Bcl-2 at the ER, its role for the modulation of calcium fluxes and its influence on caspase activation at the ER are reviewed.     

Role of the carboxyl terminal stop transfer sequence of UGT1A6 membrane protein in ER targeting and translocation of upstream lumenal domain

We investigated the role of the stop transfer sequence of human UGT1A6 in ER assembly and enzyme activity. We found that this sequence was able to address and translocate the upstream lumenal domain into microsomal membranes in vitro co- and posttranslationally. The signal activity of this sequence was further demonstrated in HeLa cells by its ability to target and maintain the CD4 protein deleted from both the N-terminal signal peptide and C-terminal transmembrane domain into the ER. We showed that total or partial deletion of the stop transfer sequence of UGT1A6 severely impaired enzyme activity highlighting its importance in both membrane assembly and function.     

Aberrant membrane insertion of a cytoplasmic tail deletion mutant of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a type II glycoprotein oriented in the plasma membrane with its amino terminus in the cytoplasm and its carboxy terminus external to the cell. We have previously shown that the membrane insertion of HN protein requires signal recognition particle SRP, occurs cotranslationally, and utilizes the same GTP-dependent step that has been described for secretory proteins, type I proteins, and multispanning proteins (C. Wilson, R. Gilmore, and T. Morrison, Mol. Cell. Biol. 7:1386-1392, 1987; C. Wilson, T. Connolly, T. Morrison, and R. Gilmore, J. Cell Biol. 107:69-77, 1988). The role of the amino-terminal cytoplasmic domain in the faithful membrane insertion of this type II protein was explored by characterizing the membrane integration of a mutant lacking 23 of the 26 amino acids of the cytoplasmic domain. The mutant protein was able to interact with SRP, resulting in translation inhibition, membrane targeting, and membrane translocation, but the efficiency of translocation was considerably lower than for the wild-type HN protein. In addition, a significant proportion of the mutant protein synthesized in the presence of SRP and microsomal membranes was associated with the membrane in an EDTA- and alkali-insensitive manner yet integrated into membranes with its carboxy-terminal domain on the cytoplasmic side of membrane vesicles. Membrane-integrated molecules with this reverse orientation were not detected when the mutant protein was synthesized in the absence of SRP or a functional SRP receptor. Truncated mRNAs encoding amino-terminal segments of the wild-type and mutant proteins were translated to prepare ribosomes bearing arrested nascent chains. The arrested mutant nascent chain, in contrast to the wild-type nascent chain, was also able to insert into membranes in a GTP- and SRP-independent manner. Results suggest that the cytoplasmic domain plays a role in the proper membrane insertion of this type II glycoprotein.     

Membrane assembly of the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred orientation.

The M protein of mouse hepatitis virus strain A59 is a triple-spanning membrane protein which assembles with an uncleaved internal signal sequence, adopting an NexoCcyt orientation. To study the insertion mechanism of this protein, domains potentially involved in topogenesis were deleted and the effects analyzed in topogenesis were deleted and the effects analyzed in several ways. Mutant proteins were synthesized in a cell-free translation system in the presence of microsomal membranes, and their integration and topology were determined by alkaline extraction and by protease-protection experiments. By expression in COS-1 and Madin-Darby canine kidney-II cells, the topology of the mutant proteins was also analyzed in vivo. Glycosylation was used as a biochemical marker to assess the disposition of the NH2 terminus. An indirect immunofluorescence assay on semi-intact Madin-Darby canine kidney-II cells using domain-specific antibodies served to identify the cytoplasmically exposed domains. The results show that each membrane-spanning domain acts independently as an insertion and anchor signal and adopts an intrinsic preferred orientation in the lipid bilayer which corresponds to the disposition of the transmembrane domain in the wild-type assembled protein. These observations provide further insight into the mechanism of membrane integration of multispanning proteins. A model for the insertion of the coronavirus M protein is proposed.     

The N-terminal beta-barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies.

Phospholipase A2 and a particular isoform of lipoxygenase are synthesized and transferred to lipid bodies during the stage of triacylglycerol mobilization in germinating cucumber seedlings. Lipid body lipoxygenase (LBLOX) is post-translationally transported to lipid bodies without proteolytic modification. Fractionation of homogenates from cucumber cotyledons or transgenic tobacco leaves expressing LBLOX showed that a small but significant amount was detectable in the microsomal fraction. A beta-barrel-forming N-terminal domain in the structure of LBLOX, as deduced from sequence data, was shown to be crucial for selective intracellular transport from the cytosol to lipid bodies. Although a specific signal sequence for targeting protein domains to the lipid bodies could not be established, it was evident that the beta-barrel represents a membrane-binding domain that is functionally comparable with the C2 domains of mammalian phospholipases. The intact beta-barrel of LBLOX was demonstrated to be sufficient to target in vitro a fusion protein of LBLOX beta-barrel with glutathione S-transferase (GST) to lipid bodies. In addition, binding experiments on liposomes using lipoxygenase isoforms, LBLOX deletions and the GST-fusion protein confirmed the role of the beta-barrel as the membrane-targeting domain. In this respect, the cucumber LBLOX differs from cytosolic isoforms in cucumber and from the soybean LOX-1. When the beta-barrel of LBLOX was destroyed by insertion of an additional peptide sequence, its ability to target proteins to membranes was abolished.