Amino-terminal Transmembrane Domain Research Articles

Topology and Function of the S. cerevisiae Autophagy Protein Atg15.

The putative phospholipase Atg15 is required for the intravacuolar lysis of autophagic bodies and MVB vesicles. Intracellular membrane lysis is a highly sophisticated mechanism that is not fully understood. The amino-terminal transmembrane domain of Atg15 contains the sorting signal for entry into the MVB pathway. By replacing this domain, we generated chimeras located in the cytosol, the vacuole membrane, and the lumen. The variants at the vacuole membrane and in the lumen were highly active. Together with the absence of Atg15 from the phagophore and autophagic bodies, this suggests that, within the vacuole, Atg15 can lyse vesicles where it is not embedded. In-depth topological analyses showed that Atg15 is a single membrane-spanning protein with the amino-terminus in the cytosol and the rest, including the active site motif, in the ER lumen. Remarkably, only membrane-embedded Atg15 variants affected growth when overexpressed. The growth defects depended on its active site serine 332, showing that it was linked to the enzymatic activity of Atg15. Interestingly, the growth defects were independent of vacuolar proteinase A and vacuolar acidification.

From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle.

The TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). In Escherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them. Canonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport across the outer membrane for nutrients that are too large, too scarce, or too important for diffusion-limited transport. A proton gradient across the cytoplasmic membrane is converted by a multiprotein complex into mechanical energy that drives high-affinity active transport across the outer membrane. This system is also of interest since one of its uses in pathogenic bacteria is for competition with the host for the essential element iron. Understanding the mechanism of the TonB system will allow design of antibiotics targeting iron acquisition.

Membrane anchoring of Epstein-Barr virus gp42 inhibits fusion with B cells even with increased flexibility allowed by engineered spacers.

ABSTRACTWe recently described the architecture of the Epstein-Barr virus (EBV) fusion-triggering complex consisting of the EBV B cell receptor human leukocyte antigen (HLA) class II and the EBV-encoded proteins gp42 and gH/gL. The architecture of this structure positioned the main body of gp42, comprising the C-type lectin domain (CTLD), away from the membrane and distant from where the membrane-bound form of gp42 might be tethered. gp42 is a type II membrane glycoprotein, with functional gp42 formed by cleavage near the gp42 amino-terminal transmembrane domain. This cleavage results in an approximately 50-amino-acid unstructured region that is responsible for binding gH/gL with nanomolar affinity. Our previous studies had shown that membrane-bound gp42 is not functional in B cell fusion. To investigate whether we could restore gp42 function by extending it from the membrane, we introduced one, two, and four structured immunoglobulin-like domains from muscle protein titin into a membrane-bound form of gp42 and tested function in binding to gHgL and HLA class II and function in fusion. We hypothesized that cleavage of gp42 generates a soluble functional form that relieves steric hindrance imposed on gHgL by membrane-bound gp42. All of the linker mutants had a dominant-negative effect on gp42 function, indicating that gp42 fusion function could not be restored simply by the addition of one to four titin domains.

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.

Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice.

Plant chitinases, a class of glycosyl hydrolases, participate in various aspects of normal plant growth and development, including cell wall metabolism and disease resistance. The rice (Oryza sativa) genome encodes 37 putative chitinases and chitinase-like proteins. However, none of them has been characterized at the genetic level. In this study, we report the isolation of a brittle culm mutant, bc15, and the map-based cloning of the BC15/OsCTL1 (for chitinase-like1) gene affected in the mutant. The gene encodes the rice chitinase-like protein BC15/OsCTL1. Mutation of BC15/OsCTL1 causes reduced cellulose content and mechanical strength without obvious alterations in plant growth. Bioinformatic analyses indicated that BC15/OsCTL1 is a class II chitinase-like protein that is devoid of both an amino-terminal cysteine-rich domain and the chitinase activity motif H-E-T-T but possesses an amino-terminal transmembrane domain. Biochemical assays demonstrated that BC15/OsCTL1 is a Golgi-localized type II membrane protein that lacks classical chitinase activity. Quantitative real-time polymerase chain reaction and β-glucuronidase activity analyses indicated that BC15/OsCTL1 is ubiquitously expressed. Investigation of the global expression profile of wild-type and bc15 plants, using Illumina RNA sequencing, further suggested a possible mechanism by which BC15/OsCTL1 mediates cellulose biosynthesis and cell wall remodeling. Our findings provide genetic evidence of a role for plant chitinases in cellulose biosynthesis in rice, which appears to differ from their roles as revealed by analysis of Arabidopsis (Arabidopsis thaliana).

Role of the amino-terminal transmembrane domain of sulfonylurea receptor SUR2B for coupling to KIR6.2, ligand binding, and oligomerization

ATP-sensitive K(+) (K(ATP)) channels consist of two types of subunits, K(IR)6.x that form the pore, and sulfonylurea receptors (SURs) that serve as regulatory subunits. SURs are ATP-binding cassette (ABC) proteins and contain, in addition to two nucleotide binding folds, the binding sites for channel openers such as diazoxide and P1075 and channel inhibitors such as glibenclamide (GBC) and repaglinide. Structurally, SURs differ from most eukaryotic ABC proteins by an additional amino-terminal transmembrane domain (TMD0); in case of SUR1, the subunit of the pancreatic K(ATP) channel, TMD0 serves as a major domain for association with K(IR). In this study we sought to elucidate the roles of TMD0 in SUR2B, the smooth muscle gating subunit, in the coupling between SUR2B and K(IR)6.2, in the self-association of SUR2B and in channel modulator binding to SUR2B. SUR2B has a weaker affinity for sulfonylureas thus SUR2B(Y1206S), with a higher affinity for GBC, but an equivalent opener binding was used. Association of SUR2B(YS)Δ, lacking TMD0, with K(IR)6.2 was shown by immunoprecipitation; however, no evidence for formation of functional channels was obtained. SUR2B(YS)Δ self-associates like SUR2B(YS) and binds GBC, repaglinide, and P1075 with slightly reduced affinities. The binding profile of the SUR2B(YS)Δ/K(IR)6.2 complex differs slightly but significantly from that of SUR2B(YS)Δ alone showing impaired allosteric coupling of binding sites. We conclude that TMD0 is not required for oligomerization of SUR2B, is of only minor importance in ligand binding, but is essential for both functional and allosteric coupling of SUR2B to K(IR)6.2.

The Pepper E3 Ubiquitin Ligase RING1 Gene,CaRING1, Is Required for Cell Death and the Salicylic Acid-Dependent Defense Response

Ubiquitination is essential for ubiquitin/proteasome-mediated protein degradation in plant development and defense. Here, we identified a novel E3 ubiquitin ligase RING1 gene, CaRING1, from pepper (Capsicum annuum). In pepper, CaRING1 expression is induced by avirulent Xanthomonas campestris pv vesicatoria infection. CaRING1 contains an amino-terminal transmembrane domain and a carboxyl-terminal RING domain. In addition, it displays in vitro E3 ubiquitin ligase activity, and the RING domain is essential for E3 ubiquitin ligase activity in CaRING1. CaRING1 also localizes to the plasma membrane. In pepper plants, virus-induced gene silencing of CaRING1 confers enhanced susceptibility to avirulent X. campestris pv vesicatoria infection, which is accompanied by compromised hypersensitive cell death, reduced expression of PATHOGENESIS-RELATED1, and lowered salicylic acid levels in leaves. Transient expression of CaRING1 in pepper leaves induces cell death and the defense response that requires the E3 ubiquitin ligase activity of CaRING1. By contrast, overexpression of CaRING1 in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to hemibiotrophic Pseudomonas syringae pv tomato and biotrophic Hyaloperonospora arabidopsidis infections. Taken together, these results suggest that CaRING1 is involved in the induction of cell death and the regulation of ubiquitination during the defense response to microbial pathogens.

Depletion of the Membrane-Associated Acyl-Coenzyme A-Binding Protein ACBP1 Enhances the Ability of Cold Acclimation in Arabidopsis

In Arabidopsis (Arabidopsis thaliana), a family of six genes encodes acyl-coenzyme A-binding proteins (ACBPs). A member of this family, ACBP1, contains an amino-terminal transmembrane domain that targets it to the plasma membrane and the endoplasmic reticulum. To investigate ACBP1 function, ACBP1-overexpressing transgenic Arabidopsis plants were characterized using lipid analysis. ACBP1 overexpressors showed reduction in several species of diunsaturated phosphatidylcholine (PC), prompting us to investigate if they were altered in response to freezing stress. ACBP1 overexpressors demonstrated increased freezing sensitivity accompanied by a decrease in PC and an increase in phosphatidic acid (PA), while acbp1 mutant plants showed enhanced freezing tolerance associated with PC accumulation and PA reduction. We also showed binding of a recombinant eukaryotic ACBP (ACBP1) to PA, indicative of the possibility of enhanced PA interaction in ACBP1 overexpressors. Since phospholipase Dalpha1 (PLDalpha1) is a major enzyme promoting the hydrolysis of PC to PA, PLDalpha1 expression was examined and was observed to be higher in ACBP1 overexpressors than in acbp1 mutant plants. In contrast, the expression of PLDdelta, which plays a positive role in freezing tolerance, declined in the ACBP1 overexpressors but increased in acbp1 mutant plants. Given that ACBP1 is localized to the endoplasmic reticulum and plasma membrane, it may regulate the expression of PLDalpha1 and PLDdelta by maintaining a membrane-associated PA pool through its ability to bind PA. Moreover, both genotypes showed no alterations in proline and soluble sugar content or in cold-regulated (COR6.6 and COR47) gene expression, suggesting that the ACBP1-mediated response is PLD associated and is independent of osmolyte accumulation.

Functional dissection of transmembrane domains of human TAP-like (ABCB9)

An ABC transporter, TAP-Like (TAPL), was dissected into its amino-terminal transmembrane domain and the following core domain. When these domains were transiently expressed as tagged proteins with a His6- or Myc-epitope tag, the amino-terminal ones (Met 1-Lys 182) could not associate with each other, or with the full-length transporter (Met 1-Ala 766). However, both the core domain (Arg 141-Ala 766) and full-length protein mutually interacted. The amino-terminal domain (Met 1-Arg 141) as well as the full-length transporter fused with fluorescent protein GFP was sorted to lysosomal membranes upon their stable expression, as visualized by means of fluorescent microscopy, while the core domain (Arg 141-Ala 766) was broadly distributed in the intra-cellular membranes. These results suggest that the sorting signal for lysosomes is present within the amino-terminal transmembrane domain (Met 1-Arg 141) of the TAPL molecule.

Functional and Structural Characterization of 2B Viroporin Membranolytic Domains

Nonstructural 2B viroporin is an intracellularly produced pore-forming protein required for effective enteroviral and rhinoviral replication. The sequence of 2B displays two putative interconnected transmembrane domains, which are predicted to insert into the negatively charged membranes of target organelles forming an integral hairpin. The use of an overlapping peptide library that spanned the complete 2B sequence has recently allowed the mapping of the cell plasma membrane porating activity to the partially amphipathic, amino-terminal transmembrane domain (TM1, residues 35-55). We describe here that although the TM1 peptide was effective in permeabilizing uncharged membranes, it induced marginal lysis of anionic bilayers. In fact, only the peptide representing the highly conserved carboxy-terminal transmembrane domain (TM2, residues 59-82) reproduced the capacity of the full 2B protein to efficiently permeabilize bilayers made of anionic phospholipids. Insertion into lipid monolayers and circular dichroism determinations were, however, consistent with penetration of the TM1 helix into both anionic and zwitterionic membranes, while TM2 interacting with membranes assumed a mixture of conformations. Moreover, addition of TM1 strongly stimulated TM2-induced permeabilization of the anionic membranes. In combination, TM1 and TM2 formed a complex that had structural properties, including a high proportion of extended nonhelical secondary structure, that were distinct from those of the individual peptides. Finally, a comparison of antimicrobial and hemolytic activities further underscored the TM1 domain's cytolytic character. Overall, our data support the idea that the cytolytic activity of TM1 in the negatively charged cell endomembranes targeted by 2B viroporin requires the cooperation of both transmembrane domains.

Autophosphorylation and Dephosphorylation by Soluble Forms of the Nitrate-Responsive Sensors NarX and NarQ from Escherichia coli K-12

NarX-NarL and NarQ-NarP are paralogous two-component regulatory systems that control Escherichia coli gene expression in response to the respiratory oxidants nitrate and nitrite. Nitrate stimulates the autophosphorylation rates of the NarX and NarQ sensors, which then phosphorylate the response regulators NarL and NarP to activate and repress target operon transcription. Here, we investigated both the autophosphorylation and dephosphorylation of soluble sensors in which the maltose binding protein (MBP) has replaced the amino-terminal transmembrane sensory domain. The apparent affinities (K(m)) for ADP were similar for both proteins, about 2 microM, whereas the affinity of MBP-NarQ for ATP was lower, about 23 microM. At a saturating concentration of ATP, the rate constant of MBP-NarX autophosphorylation (about 0.5 x 10(-4) s(-1)) was lower than that observed for MBP-NarQ (about 2.2 x 10(-4) s(-1)). At a saturating concentration of ADP, the rate constant of dephosphorylation was higher than that of autophosphorylation, about 0.03 s(-1) for MBP-NarX and about 0.01 s(-1) for MBP-NarQ. For other studied sensors, the published affinities for ADP range from about 16 microM (KinA) to about 40 microM (NtrB). This suggests that only a small proportion of NarX and NarQ remain phosphorylated in the absence of nitrate, resulting in efficient response regulator dephosphorylation by the remaining unphosphorylated sensors.

Expression of the Membrane-Associated Resistance Protein RPW8 Enhances Basal Defense Against Biotrophic Pathogens

The powdery mildew resistance genes RPW8.1 and RPW8.2 from Arabidopsis differ from the other isolated plant resistance (R) genes in their predicted protein domains and their resistance spectrum. The two homologous RPW8 genes encode small proteins featuring a predicted amino-terminal transmembrane anchor domain and a coiled-coil domain and confer resistance to a broad spectrum of powdery mildews. Here, we show that Arabidopsis plants expressing the RPW8 genes have enhanced resistance to another biotrophic pathogen, Hyaloperonospora parasitica, raising the possibility that the RPW8 genes may function to enhance salicylic-acid-dependent basal defenses, rather than as powdery-mildew-specific R genes. When overexpressed from their native promoters, the RPW8 genes confer enhanced resistance to the Cauliflower mosaic virus, but render plants more susceptible to the necrotrophic fungal pathogens Alternaria and Botrytis spp. Furthermore, we show that the RPW8 proteins appear to be localized to the endomembrane system, overlapping with the endoplasmic reticulum-associated small GTPase SAR1, and accumulate to higher levels in response to application of exogenous salicylic acid, one of the signaling molecules of plant defense.

Cell-based assay for β-secretase activity

The cerebral deposition of amyloid β-peptide (Aβ) is a major factor in the etiology of Alzheimer’s disease. β-Secretase (BACE) initiates the generation of Aβ by cleaving the amyloid precursor protein at the β-site and is therefore a prime target for therapeutic intervention. Here we report a cell-based method suitable for monitoring BACE activity and the efficacy of protease inhibitors. A fusion protein containing the amino-terminal transmembrane domain of Golgi α-mannosidase II, a Drosophila Golgi integral membrane protein, linked to human alkaline phosphatase (AP) by a short β-site sequence, was expressed in Drosophila S2 cells. While the uncleaved fusion protein was retained in the Golgi apparatus, cleavage of the β-site by BACE resulted in the release of AP to the culture medium, where it was easily detected and quantified. Three peptidomimetic inhibitors (LB83190, LB83192, LB83202) were tested for their efficacy with this cell-based assay. While LB83190 and LB83192 effectively blocked BACE activity, LB83202, a carboxylated derivative of LB83192, did not. This is consistent with the inability of LB83202 to permeate the cell membrane. The present cell-based assay could provide a convenient tool for high-throughput screening of substances that can interfere with BACE in living cells.

Selective Trafficking of Non-Cell-Autonomous Proteins Mediated by NtNCAPP1

In plants, cell-to-cell communication is mediated by plasmodesmata and involves the trafficking of non-cell-autonomous proteins (NCAPs). A component in this pathway, Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (NtNCAPP1), was affinity purified and cloned. Protein overlay assays and in vivo studies showed that NtNCAPP1 is located on the endoplasmic reticulum at the cell periphery and displays specificity in its interaction with NCAPs. Deletion of the NtNCAPP1 amino-terminal transmembrane domain produced a dominant-negative mutant that blocked the trafficking of specific NCAPs. Transgenic tobacco plants expressing this mutant form of NtNCAPP1 and plants in which the NtNCAPP1 gene was silenced were compromised in their ability to regulate leaf and floral development. These results support a model in which NCAP delivery to plasmodesmata is both selective and regulated.

Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer.

Dysregulated proteolysis is a hallmark of cancer. Malignant cells require a range of proteolytic activities to enable growth, survival, and expansion. Serine proteases of the S1 or trypsin-like family have well recognized roles in the maintenance of normal homeostasis as well as in the pathology of diseases such as cancer. Recently a rapidly expanding subgroup of S1 proteases has been recognized that are directly anchored to plasma membranes. These membrane anchored serine proteases are anchored either via a carboxy-terminal transmembrane domain (Type I), a carboxy terminal hydrophobic region that functions as a signal for membrane attachment via a glycosyl-phosphatidylinositol linkage (GPI-anchored), or via an amino terminal proximal transmembrane domain (Type II or TTSP). The TTSPs also encode multiple domains in their stem regions that may function in regulatory interactions. The serine protease catalytic domains of these enzymes show high homology but also possess features indicating unique substrate specificities. It is likely that the membrane anchored serine proteases have evolved to perform complex functions in the regulation of cellular signaling events at the plasma membrane and within the extracellular matrix. Disruption or mutation of several of the genes encoding these proteases are associated with disease. Many of the membrane anchored serine proteases show restricted tissue distribution in normal cells, but their expression is widely dysregulated during tumor growth and progression. Diagnostic or therapeutic targeting of the membrane anchored serine proteases has potential as promising new approaches for the treatment of cancer and other diseases.

Identification, Genomic Organization, and mRNA Expression of LACTB, Encoding a Serine β-Lactamase-like Protein with an Amino-terminal Transmembrane Domain

Database searching with bacterial serine β-lactamases identified mouse expressed sequence tags (ESTs) with significant similarity scores.The cloned mouse cDNA encodes a novel 551-amino-acid protein, LACTB, with a predicted amino-terminal transmembrane domain but no signal peptide. It contains an active site motif related to C-class β-lactamases. Homologues were detected in sequence data from human, rat, cow, rabbit, pig, toad, zebrafish, and Caenorhabditis elegans, but not in Saccharomyces cerevisae or Drosophila melanogaster. The genes were mapped to human chromosome 15q22.1 and mouse chromosome 9. Sequencing of a 14.7-kb fragment of mouse genomic DNA defined six exons. A virtual human cDNA and a 549-residue protein, predicted from unfinished genomic sequence, showed the same intron/exon structure. Northern blot analysis showed expression of the 2.3-kb mRNA predominantly in mouse liver and human skeletal muscle. This is the first reported vertebrate example of this microbial peptidase family.

Nucleotide-dependent binding of the GTPase domain of the signal recognition particle receptor beta-subunit to the alpha-subunit.

The signal recognition particle (SRP) receptor (SR) is a heterodimer of two polypeptides (SRalpha and SRbeta) that each contain a GTP-binding domain. The GTP-binding domain in the peripheral membrane SRalpha subunit has a well defined role in regulating targeting of SRP-ribosome-nascent chain complexes to the translocon. The only well established function for the transmembrane SRbeta subunit is anchoring SRalpha on the endoplasmic reticulum membrane. Deletion of the amino-terminal transmembrane domain of SRbeta did not affect receptor dimerization, but revealed a cryptic translocation signal that overlaps the GTPase domain. We demonstrate that the domain of SRalpha that binds SRbeta does so by binding directly to the nucleotide-bound form of the GTPase domain of SRbeta. An SRbeta mutant containing an amino acid substitution that allows the GTPase domain to bind XTP dimerized with SRalpha most efficiently in the presence of XTP or XDP, but not ATP. Our results suggest an additional level of regulation of SRP receptor function based on regulated dissociation of the receptor subunits.

Membrane topology of the NixA nickel transporter of Helicobacter pylori: two nickel transport-specific motifs within transmembrane helices II and III.

NixA, the high-affinity cytoplasmic membrane nickel transport protein of Helicobacter pylori, imports Ni(2+) into the cell for insertion into the active site of the urease metalloenzyme, which is required for gastric colonization. NixA fractionates with the cytoplasmic membrane, and protein cross-linking studies suggest that NixA functions as a monomer. A preliminary topological model of NixA with seven transmembrane domains was previously proposed based on hydropathy, charge dispersion, and homology to other transporters. To test the proposed topology of NixA and relate critical residues to specific structural elements, a series of 21 NixA-LacZ and 21 NixA-PhoA fusions were created along the entire length of the protein. Expression of reporter fusions was confirmed by Western blotting with beta-galactosidase- and alkaline phosphatase-specific antisera. The activities of reporter fusions near to and upstream of the predicted translational initiation demonstrated the presence of an additional amino-terminal transmembrane domain including a membrane localization signal. Activities of fusions immediately adjacent to motifs which have been shown to be requisite for Ni(2+) transport localized these motifs entirely within transmembrane domains II and III. Fusion activities localized six additional Asp and Glu residues which reduced Ni(2+) transport by >90% when mutated within or immediately adjacent to transmembrane domains II, V, VI, and VII. All fusions strongly support a model of NixA in which the amino and carboxy termini are located in the cytoplasm and the protein possesses eight transmembrane domains.

Association of Protein Kinase Cμ with Type II Phosphatidylinositol 4-Kinase and Type I Phosphatidylinositol-4-phosphate 5-Kinase

Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.