Putative Membrane-spanning Segments Research Articles

TRIC channels supporting efficient Ca2+ release from intracellular stores

Trimeric intracellular cation-selective (TRIC) channel subtypes, namely TRIC-A and TRIC-B, are derived from distinct genes and distributed throughout the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes. TRIC-A is preferentially expressed at high levels in excitable tissues, while TRIC-B is ubiquitously detected at relatively low levels in various tissues. TRIC channels are composed of ~300 amino acid residues and contain three putative membrane-spanning segments to form a bullet-shaped homo-trimeric assembly. Both native and purified recombinant TRIC subtypes form functional monovalent cation-selective channels in a lipid bilayer reconstitution system. The electrophysiological data indicate that TRIC channels behave as K(+) channels under intracellular conditions, although the detailed channel characteristics remain to be investigated. The pathophysiological defects detected in knockout mice suggest that TRIC channels support SR/ER Ca(2+) release mediated by ryanodine (RyR) and inositol trisphosphate receptor (IP(3)R) channels. For example, Tric-a-knockout mice develop hypertension resulting from vascular hypertonicity, and the mutant vascular smooth muscle cells exhibit insufficient RyR-mediated Ca(2+) release for inducing hyperpolarization. Tric-b-knockout mice show respiratory failure at birth, and IP(3)R-mediated Ca(2+) release essential for surfactant handling is impaired in the mutant alveolar epithelial cells. Moreover, double-knockout mice lacking both TRIC subtypes show embryonic heart failure, and SR Ca(2+) handling is deranged in the mutant cardiomyocytes. Current evidence strongly suggests that TRIC channels mediate counter-K(+) movements, in part, to facilitate physiological Ca(2+) release from intracellular stores.

The Structure and Interactions of SpoIISA and SpoIISB, a Toxin-Antitoxin System in Bacillus subtilis

Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase σ factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, σF. Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 Å spacing, revealing a CSpoIISA2·SpoIISB2 heterotetramer. CSpoIISA has a single domain α/β structure resembling a GAF domain with an extended α-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA·SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed.

Distinct Structural Elements in the First Membrane-spanning Segment of the Epithelial Sodium Channel

Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an alpha2betagamma or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate site within alphaM1. We performed site-directed mutagenesis at this site and found that, although the mutants reduced channel activity, ENaC protein expression at the plasma membrane was unaffected. To deduce the role of alphaM1 in the pore structure of ENaC, we performed tryptophan-scanning mutagenesis throughout alphaM1 (residues 110-130). We found that mutations within the amino-terminal part of alphaM1 had effects on activity and selectivity with a periodicity consistent with a helical structure but no effect on channel surface expression. We also observed that mutations within the carboxyl-terminal part of alphaM1 had effects on activity and selectivity but with no apparent periodicity. Additionally, these mutants reduced channel surface expression. Our data support a model in which the amino-terminal half of alphaM1 is alpha-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of alphaM1 may be helical or assume a different conformation and may be involved in tertiary interactions essential to proper channel folding or assembly. Together, our data suggest that alphaM1 is divided into two distinct regions.

A receptor-like kinase from Arabidopsis thaliana is a calmodulin-binding protein.

Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways.

Role of the CD47-SHPS-1 system in regulation of cell migration.

SHPS-1 is a transmembrane protein whose extracellular region interacts with CD47 and whose cytoplasmic region undergoes tyrosine phosphorylation and there by binds the protein tyrosine phosphatase SHP-2. Formation of this complex is implicated in regulation of cell migration by an unknown mechanism. A CD47-Fc fusion protein or antibodies to SHPS-1 inhibited migration of human melanoma cells or of CHO cells overexpressing SHPS-1. Overexpression of wild-type SHPS-1 promoted CHO cell migration, whereas expression of the SHPS-1-4F mutant, which lacks the phosphorylation sites required for SHP-2 binding, had no effect. Antibodies to SHPS-1 failed to inhibit migration of CHO cells expressing SHPS-1-4F. SHPS-1 ligands induced the dephosphorylation of SHPS-1 and dissociation of SHP-2. Antibodies to SHPS-1 also enhanced Rho activity and induced both formation of stress fibers and adoption of a less polarized morphology in melanoma cells. Our results suggest that engagement of SHPS-1 by CD47 prevents the positive regulation of cell migration by this protein. The CD47- SHPS-1 system and SHP-2 might thus contribute to the inhibition of cell migration by cell-cell contact.

On the Accuracy of Transmembrane Segment Prediction of Helical Integral Membrane Proteins

Integral membrane proteins play a vital role in a number of essential biological functions. Although abundant, about 30% of genes are known to code for membrane proteins, the number of solved structures in the pdb is less than 1%. Thus, structure prediction of membrane proteins is an essential tool for understanding their functions. A fundamental characteristic of the predicted structure is the topology – identification of trans-membrane segments and the overall orientation with respect to the membrane (intraor extra-cellular). Several prediction methods have been developed for this purpose, both knowledge-based and residue hydrophobicity-based. Although the performances of almost all of these methods are rather high, short loops and long helices are predicted less accurately [1]. One of the problems of estimating accuracy of different prediction methods is the absence of experimentally reliable trans-membrane annotations to compare with. Thus, one is forced to compare prediction versus prediction, where the assumed transmembrane segments typically are identical to entire helix lengths of putative membrane-spanning segments in the pdb structure. Adding hydrophobicity information to the structural data, and by optimizing the resulting membrane-protein mismatch energy, we provide alternate assignments of trans-membrane segments of membrane proteins whose structures are known. This new assignment is used to revaluate the prediction accuracy of established prediction methods and hydrophobicity-based methods. The implications of our results are discussed.

Mutations in Flavobacterium johnsoniae gldF and gldG disrupt gliding motility and interfere with membrane localization of GldA.

Flavobacterium johnsoniae moves rapidly over surfaces by a process known as gliding motility. The mechanism of this form of motility is not known. Four genes that are required for F. johnsoniae gliding motility, gldA, gldB, gldD, and ftsX, have recently been described. GldA is similar to the ATP-hydrolyzing components of ATP binding cassette (ABC) transporters. Tn4351 mutagenesis was used to identify two additional genes, gldF and gldG, that are required for cell movement. gldF and gldG appear to constitute an operon, and a Tn4351 insertion in gldF was polar on gldG. pMK314, which carries the entire gldFG region, restored motility to each of the gldF and gldG mutants. pMK321, which expresses GldG but not GldF, restored motility to each of the gldG mutants but did not complement the gldF mutant. GldF has six putative membrane-spanning segments and is similar in sequence to channel-forming components of ABC transporters. GldG is similar to putative accessory proteins of ABC transporters. It has two apparent membrane-spanning helices, one near the amino terminus and one near the carboxy terminus, and a large intervening loop that is predicted to reside in the periplasm. GldF and GldG are involved in membrane localization of GldA, suggesting that GldA, GldF, and GldG may interact to form a transporter. F. johnsoniae gldA is not closely linked to gldFG, but the gldA, gldF, and gldG homologs of the distantly related gliding bacterium Cytophaga hutchinsonii are arranged in what appears to be an operon. The exact roles of F. johnsoniae GldA, GldF, and GldG in gliding are not known. Sequence similarities of GldA to components of other ABC transporters suggest that the Gld transporter may be involved in export of some material to the periplasm, outer membrane, or beyond.

Yeast V-ATPase Complexes Containing Different Isoforms of the 100-kDa a-subunit Differ in Coupling Efficiency and in VivoDissociation

The 100 kDa a-subunit of the yeast vacuolar (H(+))-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1. These genes encode unique isoforms of the a-subunit that have previously been shown to reside in different intracellular compartments in yeast. Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes. To compare the properties of V-ATPases containing Vph1p or Stv1p, Stv1p was expressed at higher than normal levels in a strain disrupted in both genes, under which conditions V-ATPase complexes containing Stv1p appear in the vacuole. Complexes containing Stv1p showed lower assembly with the peripheral V(1) domain than did complexes containing Vph1p. When corrected for this lower degree of assembly, however, V-ATPase complexes containing Vph1p and Stv1p had similar kinetic properties. Both exhibited a K(m) for ATP of about 250 microm, and both showed resistance to sodium azide and vanadate and sensitivity to nanomolar concentrations of concanamycin A. Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes. We also compared the ability of V-ATPase complexes containing Vph1p or Stv1p to undergo in vivo dissociation in response to glucose depletion. Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not. Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed glucose-dependent dissociation. Blocking delivery of Vph1p-containing complexes to the vacuole in vps21Delta and vps27Delta strains caused partial inhibition of glucose-dependent dissociation. These results suggest that dissociation of the V-ATPase complex in vivo is controlled both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.

Putative membrane components of signal transduction pathways for ambient pH regulation in Aspergillus and meiosis in Saccharomyces are homologous Addendum.

The zinc finger regions of the Aspergillus nidulans PacC transcription factor, mediating regulation of gene expression by ambient pH, and the Saccharomyces cerevisiae Rim1p transcription factor, mediating control of meiosis and invasiveness, are homologous and both transcription factors undergo proteolytic processing of the C-terminus for conversion to the functional form. In both cases, functioning of a signal transduction pathway involving several gene products is a necessary prerequisite for processing. We now show that the Aspergillus PalI pH signal transduction component is homologous to the Saccharomyces Rim9p meiotic signal transduction component throughout a region containing four hydrophobic, putative membrane-spanning segments. This suggests that PalI might be a membrane sensor for ambient pH. Deletion of the palI gene established that the less extreme phenotype of palI mutations compared with mutations in the other five genes of the pH signalling pathway is a general feature of palI mutations.

Transmembrane Topology of the Secretory Na+-K+-2Cl− Cotransporter NKCC1 Studied by in Vitro Translation

The secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is a member of a small gene family of electroneutral salt transporters. Hydropathy analyses indicate that all of these transporters have a similar general structure consisting of large hydrophilic N and C termini on either side of a central, relatively well conserved, hydrophobic domain. Programs that predict the transmembrane topology of polytopic membrane proteins identify 10-12 putative membrane-spanning segments (MSSs) in this hydrophobic domain; but to date, there is little experimental data on the structure of this region for any of these transporters. In this report, we have studied the transmembrane topology of NKCC1 using an in vitro translation system designed to test the membrane insertion properties of putative MSSs (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919). Fusion proteins consisting of putative NKCC1 MSSs inserted either (i) between an N-terminal cytosolic anchor sequence and a C-terminal reporter sequence containing multiple N-linked glycosidation sites or (ii) between an N-terminal signal anchor sequence and the same glycosidation flag were expressed in the presence of canine pancreatic microsomes. The glycosidation status of the reporter sequence, which indicated its luminal or extraluminal location in the microsomes, was then used to characterize the signal anchor or stop transfer activity of the inserted MSSs. The results of this experimental analysis yielded a topology scheme consisting of 12 membrane-spanning segments, two pairs of which apparently form rather tight hairpin-like structures within the membrane.

Rat homolog of sulfonylurea receptor 2B determines glibenclamide sensitivity of ROMK2 in Xenopus laevis oocyte.

Recent studies showed that coexpression of Kir6.1 or Kir6.2 with the sulfonylurea receptor (SUR1, SUR2A, or SUR2B) reconstituted an inwardly rectifying, ATP-sensitive K(+) channel that was inhibited by glibenclamide (2, 15-17). Here we report the isolation of a rat homolog of mouse SUR2B (denoted rSUR2B) from a rat kidney cDNA library. The rSUR2B sequence contains a 4,635-bp open reading frame that encodes a 1,545-amino acid polypeptide, showing 67% shared identity with SUR1 (a pancreatic beta-cell isoform) and 98% with both SUR2A (a brain isoform) and SUR2B (a vascular smooth muscle isoform). Consistent with the predicted structures of other members of the ATP-binding cassette (ABC) superfamily, the sequence of rSUR2B contains 17 putative membrane-spanning segments. Also, predicted Walker A and B consensus binding motifs, present in other ABC members, are conserved in the rSUR2B sequence. RT-PCR revealed that rSUR2B is widely expressed in various rat tissues including brain, colon, heart, kidney, liver, skeletal muscle, and spleen. The intrarenal distribution of the rSUR2B transcript was investigated using RT-PCR and Southern blot of microdissected tubules. The rSUR2B transcript was detected in proximal tubule, cortical thick ascending limb, distal collecting tubule, cortical collecting duct, and outer medullary collecting duct, but not medullary thick ascending limb. This distal distribution overlaps with that of ROMK. Coexpression of rSUR2B with ROMK2 cRNA (in 1:10 ratio) in Xenopus laevis oocytes resulted in whole cell Ba(2+)-sensitive K(+) currents that were inhibited by glibenclamide (50% inhibition with 0.2 mM glibenclamide). In contrast, rSUR2B did not confer significant glibenclamide sensitivity to oocytes coinjected with ROMK1 or ROMK3. The interaction between ROMK2 and rSUR2B was further studied by coimmunoprecipitation of in vitro translated rSUR2B and ROMK2. In agreement with the functional data, the rSUR2B protein was coimmunoprecipitated with ROMK2 in the ROMK2-rSUR2B cotranslated samples. Our data demonstrate that ROMK2, but not ROMK1 and ROMK3, can interact with rSUR2B to confer a sulfonylurea-sensitive K(+) channel, implicating SUR proteins in forming and regulating renal ATP-sensitive K(+) channels. The ROMK isoform specificity of glibenclamide effects suggests that the NH(2) terminus of the ROMK protein mediates rSUR2B-ROMK2 interactions.

Molecular Mechanism of Detectable Catalase-Containing Particles, Peroxisomes, in Fibroblasts from a PEX2-Defective Patient

Patients with peroxisome biogenesis disorders (PBD) can be identified by detection of peroxisomes in their fibroblasts, by means of immunocytochemical staining using an anti-catalase antibody. We report here data on three PBD patients with newly identified mutations (del550C and del642G) in the PEX2 gene which encodes a 35-kDa peroxisomal membrane protein containing two membrane-spanning and a C-terminal cysteine-rich region. Some of the fibroblasts from the patient with the del642G mutation contained numerous catalase-containing particles, whereas no fibroblasts containing such particles were found in the patient with the del550C mutation. We confirmed that the del642G mutation caused a partial defect in peroxisome synthesis and import by expression of the mutated PEX2 into PEX2-defective CHO mutant cells. We propose that the two putative membrane-spanning segments in Pex2p are important domains for peroxisome assembly and import and that a defect in one of these domains severely affects PBD patients. Furthermore, a defect in the C-terminal portion of Pex2p exposed to the cytosol containing a RING finger motif caused the mild phenotype, residual enzyme activities, and mosaic detectable peroxisomes in fibroblasts from the patient.

Channel-lining residues in the M3 membrane-spanning segment of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride-selective channel. Residues from the 12 putative membrane-spanning segments form at least part of the channel lining. We need to identify the channel-lining residues in order to understand the structural basis for the channel's functional properties. Using the substituted-cysteine-accessibility method we mutated to cysteine, one at a time, 24 consecutive residues (Asp192-Ile215) in the M3 membrane-spanning segment. Cysteines substituted for His199, Phe200, Trp202, Ile203, Pro205, Gln207, Leu211, and Leu214 reacted with charged, sulfhydryl-specific reagents that are derivatives of methanethiosulfonate (MTS). We infer that these residues are on the water-accessible surface of the protein and probably form a portion of the channel lining. When plotted on an alpha-helical wheel the exposed residues from Gln207 to Leu214 lie within an arc of 60 degrees; the exposed residues in the cytoplasmic half (His199-Ile203) lie within an arc of 160 degrees. We infer that the secondary structures of the extracellular and cytoplasmic halves of M3 are alpha-helical and that Pro205, in the middle of the M3 segment, may bend the M3 segment, moving the cytoplasmic end of the segment in toward the central axis of the channel. The bend in the M3 segment may help to narrow the channel lumen near the cytoplasmic end. In addition, unlike full-length CFTR, the current induced by the deletion construct, Delta259, is inhibited by the MTS reagents, implying that the channel structure of Delta259 is different than the channel structure of wild-type CFTR.

Molecular Characterization and Tissue Distribution of a New Organic Anion Transporter Subtype (oatp3) That Transports Thyroid Hormones and Taurocholate and Comparison with oatp2

Two complementary DNAs for the organic anion transporter subtypes oatp2 and oatp3, which transport thyroid hormones as well as taurocholate, were isolated from a rat retina cDNA library. The sequence of oatp2 is identical to that recently reported (Noé, B., Hagenbuch, B., Stieger, B., and Meier, P. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10346-10350), whereas the sequence of oatp3 is novel. oatp3 consists of 670 amino acid residues and exhibits a structural architecture common to the organic anion transporter family, possessing the 12 putative membrane-spanning segments. Oocytes injected with oatp2 and oatp3 cRNAs showed taurocholate uptake in a saturable manner. The oatp2 and oatp3 cRNA-injected oocytes also showed significant uptake of both thyroxine and triiodothyronine. Northern blot and in situ analyses showed that the oatp2 mRNA was widely expressed in neuronal cells of the central nervous system, especially in the hippocampus, cerebellum, and choroid plexus as well as in the retina and liver. The oatp3 mRNA was highly expressed in the kidney and moderately abundant in the retina. This suggests that oatp2 and oatp3 are multifunctional transporters involved in the transport of thyroid hormones in the brain, retina, liver, and kidney.

A pyrophosphate synthase gene: molecular cloning and sequencing of the cDNA encoding the inorganic pyrophosphate synthase from Rhodospirillum rubrum

The integrally membrane-bound, proton-pumping inorganic pyrophosphate (PPi) synthase in phototrophic bacteria is hitherto the only described alternative to the ATP synthase in biological electron transport phosphorylation. We have identified and sequenced the first gene coding for a pyrophosphate synthase. The deduced protein contains 660 amino acid residues and 15 putative membrane-spanning segments. It is homologous to the vacuolar pyrophosphatases from plants.

50] Overexpression, purification, and function of first nucleotide-binding fold of cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) is the most common lethal autosomal recessive genetic disease in the Caucasian population. The disease results from mutations in the gene that codes for the cystic fibrosis transmembrane conductance regulator (CFTR) protein comprised of 1480 amino acid residues. CFTR embraces two transmembrane domains, each having six putative membrane-spanning segments; two nucleotide-binding folds, NBF1 and NBF2, each possessing a Walker A and B nucleotide-binding consensus sequence, and a regulatory (R) domain having numerous acidic and basic amino acid residues as well as serine and threonine residues that can be phosphorylated by some cAMP dependent protein kinases. Mutations in CFTR occur within or near the two NBFs. Most patients with cystic fibrosis have at least one allele in which the codon for phenylalanine-508 in NBF1 is deleted. The interaction of intracellular adenosine triphosphate (ATP) with NBF1 (or NBF2) may be essential for chloride channel activation.

SARPs: a family of secreted apoptosis-related proteins.

Quiescent mouse embryonic C3H/10T1/2 cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T1/2 cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T1/2 cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, we identified and cloned a cDNA designated sarp1 (secreted apoptosis-related protein) that is expressed in quiescent but not in exponentially growing 10T1/2 cells. Hybridization studies with sarp1 revealed two additional family members. Cloning and sequencing of sarp2 and sarp3 revealed 38% and 40% sequence identity to sarp1, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with sarp1 or infected with SARP1-expressing adenovirus became more resistant, whereas cells transfected with sarp2 displayed increased sensitivity to different proapoptotic stimuli. Expression of sarp family members is tissue specific. sarp mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of beta-catenin, suggesting that SARPs interfere with the Wnt-frizzled proteins signaling pathway.

The Yeast JEM1p Is a DnaJ-like Protein of the Endoplasmic Reticulum Membrane Required for Nuclear Fusion

DnaJ-like proteins are functional partners for Hsp70 molecular chaperones. Complete nucleotide sequencing of yeast chromosome X has revealed that an open reading frame YJL073w encodes a novel member of the DnaJ-like protein family. The open reading frame represents a protein of 692 amino acids with a J-domain and one putative membrane-spanning segment. An epitope-tagged version of the protein was anchored in the endoplasmic reticulum (ER) membrane and its J-domain faced the ER lumen. We therefore propose to designate this gene JEM1 (DnaJ-like protein of the ER membrane) and to designate its gene product JEM1p. The JEM1 gene is not essential for cell growth, but double disruption of the JEM1 gene and the SCJ1 gene, which encodes another DnaJ-like protein in the ER lumen, causes growth arrest at elevated temperature. The Deltajem1 mutant is defective in nuclear fusion, karyogamy, during mating. A mutant JEM1p carrying a mutation in the highly conserved His-Pro-Asp sequence in the J-domain could not complement either temperature-sensitive growth of the Deltajem1 Deltascj1 double mutant or defects in karyogamy of the Deltajem1 mutant. JEM1p likely assists the functions of BiP, Hsp70 in the ER, including karyogamy.

Microsomal P450 2C3 Is Expressed as a Soluble Dimer in Escherichia coliFollowing Modifications of Its N-terminus

A hydrophobic segment present in the N-terminus of microsomal P450s is thought to serve as a membrane anchor. A variant of P450 2C3 was constructed, P450 2C3d, that lacked the putative membrane-spanning segment of the N-terminus, residues 3–20. This construct also incorporated substitutions of an alanine for 2Asp to facilitate expression in Escherichia coliand of serines for 24His and 25Gly to introduce a restriction site. P450 2C3d is expressed at relatively high levels in E. coli,800–1200 nmol/liter of culture medium. In contrast to P450 2C3mod, which retains a membrane-spanning N-terminal sequence modified for expression in E. coli,the subcellular distribution of P450 2C3d in E. coliis dependent on the ionic strength of the buffer used for cell disruption. In low ionic strength buffers, 2C3d was mainly localized in the membrane fraction, whereas in buffers containing 1 mNaCl or 0.5 mKPi, P450 2C3d was predominately found in the soluble fraction, indicating that deletion of the hydrophobic segment converted the intrinsic membrane protein to an extrinsic one. P450 2C3d was further modified by the incorporation of four histidine residues at the C-terminus (P450 2C3dH), and this enzyme could be purified in the absence of detergent using immobilized metal affinity chromatography following extraction from isolated membranes in high salt buffers. The catalytic properties of the purified, modified enzymes are similar to those of the native enzyme. Size-exclusion chromatography indicated that 2C3dH and 2C3d are predominantly dimers, whereas 2C3 is a larger oligomer (>8-mer). Moreover, the detergents sodium cholate and Chaps each dissociate the dimers of 2C3dH to monomers at concentrations that do not alter the aggregation state of 2C3. These modifications are likely to facilitate attempts to crystallize the catalytic domains of microsomal P450s.

Positional cloning of a gene for nematode resistance in sugar beet.

The Hs1(pro-1) locus confers resistance to the beet cyst nematode (Heterodera schachtii Schmidt), a major pest in the cultivation of sugar beet (Beta vulgaris L.). The Hs1(pro-1) gene was cloned with the use of genome-specific satellite markers and chromosomal break-point analysis. Expression of the corresponding complementary DNA in a susceptible sugar beet conferred resistance to infection with the beet cyst nematode. The native Hs1(pro-1) gene, expressed in roots, encodes a 282-amino acid protein with imperfect leucine-rich repeats and a putative membrane-spanning segment, features similar to those of disease resistance genes previously cloned from higher plants.