A maize defense-inducible gene is a major facilitator superfamily member related to bacterial multidrug resistance efflux antiporters

A defense-inducible maize gene was discovered through global mRNA profiling analysis. Its mRNA expression is induced by pathogens and defense-related conditions in various tissues involving both resistant and susceptible interactions. These include Cochliobolus heterostrophus and Cochliobolus carbonum infection, ultraviolet light treatment, the Les9 disease lesion mimic background, and plant tissues engineered to express flavonoids or the avirulence gene avrRxv. The gene was named Zm-mfs1 after it was found to encode a protein related to the major facilitator superfamily (MFS) of intregral membrane permeases. It is most closely related to the bacterial multidrug efflux protein family, typified by the Escherichia coli TetA, which are proton motive force antiporters that export antimicrobial drugs and other compounds, but which can be also involved in potassium export/proton import or potassium re-uptake. Other related plant gene sequences in maize, rice, and Arabidopsis were identified, three of which are introduced here. Among this new plant MFS subfamily, the characteristic MFS motif in cytoplasmic TM2-TM3 loop, and the antiporter family motif in transmembrane domain TM5 are both conserved, however the TM7 and the cytoplasmic TM8-TM9 loop are divergent from those of the bacterial multidrug transporters. We hypothesize that Zm-Mfs1 is a prototype of a new class of plant defense-related proteins that could be involved in either of three nonexclusive roles: (1) export of antimicrobial compounds produced by plant pathogens; (2) export of plant-generated antimicrobial compounds; and (3) potassium export and/or re-uptake, as can occur in plant defense reactions.     

A new transmembrane 4 superfamily molecule in the nematode,Caenorhabditis elegans

Transmembrane 4 superfamily (TM4SF) molecules are predominantly mammalian cell surface glycoproteins that are thought to transduce signals mediating cell development, activation, and motility. Analysis of the Genpept sequence database reveals YKK8, a novel member of the TM4SF in the nematode,Caenorhabditis elegans. YKK8 is a putative 27.4-kDa protein encoded by a gene on chromosome III of theC. elegans genome (Wilson et al. [1994]Nature 368:32–38). The assignment of YKK8 to the TM4SF is justified by three criteria: statistical comparison of protein sequences, conserved TM4SF protein sequence motifs, and conserved TM4SF intron/exon boundaries in the genomic sequence. The discovery of a TM4SF molecule in the nematode extends this superfamily to a more primitive branch of the phylogenetic tree and suggests a fundamental role for TM4SF molecules in biology. Correspondence to: M.G. Tomlinson     

Co-translational effects of temperature on membrane insertion and orientation of P-glycoprotein sequences

P-glycoprotein (pgp) is a membrane transport protein that causes multidrug resistance (MDR) by actively extruding a wide variety of cytotoxic agents out of cells. It may also function as a peptide transporter, a volume-regulated chloride channel, and an ATP channel. Previously, it has been shown that hamster pgp1 Pgp is expressed in more than one topological form and that the generation of these structures is modulated by charged amino acids flanking the predicted transmembrane (TM) segments 3 and 4 and by soluble cytoplasmic factors. Different topological structures of Pgp may be related to its different functions. In this study, we examined the effects of translation temperature on the membrane insertion process and the topologies of Pgp. Using the rabbit reticulocyte lysate expression system, we showed that translation at different temperatures affects the membrane insertion and orientation of the putative TM3 and TM4 of hamster pgp1 Pgp in a co-translational manner. This observation suggests that the membrane insertion process of TM3 and TM4 of Pgp molecules may involve a protein conducting channel and/or the interaction between TM3 and TM4, which act in a temperature sensitive manner. We speculate that manipulating temperature may provide a way to understand the structure-function relationship of Pgp and help overcome Pgp-related multidrug resistance of cancer cells.Abbreviations Pgp P-glycoprotein - MDR multidrug resistance - ABC ATP-binding cassette - RRL rabbit reticulocyte lysate - TM transmembrane - RM rough microsomes - ER endoplasmic reticulum     

Molecular and biochemical comparison of two different apyrases from Arabidopsis thaliana

Recent advances in the Arabidopsis sequencing project has elucidated the presence of two genes Atb561-A and Atb561-B that show limited homology to the DNA sequence encoding for the mammalian chromaffin granule cytochrome b-561 (cyt b-561). Detailed analysis of the structural features and conserved residues reveals, however, that the structural homology between the presumptive Arabidopsis proteins and the animal proteins is very high. All proteins are hydrophobic and show highly conserved transmembrane helices. The presumably heme-binding histidine residues in the plant and animal sequences as well as the suggested binding site for the electron acceptor, monodehydroascorbate, are strictly conserved. In contrast, the suggested electron donor (ascorbate) binding site is not very well conserved between the plant and animal sequences questioning the function of this motif. Sequence analysis of the Atb561-B gene demonstrates a different splicing than that initially predicted in silico resulting in a protein with nine extra amino acids and a significantly higher homology to the other cyt b-561 sequences. The homology between the plant and animal sequences is further supported by the strong similarity between a number of biochemical properties of the chromaffin cyt b-561 and the cyt b-561 isolated from bean hook plasma membranes. Since the mammalian cyt b-561 is considered specific to neuroadrenergic tissues, the identification of a closely related homologue in an aneural organism demonstrates that these proteins constitute a new class of widely occurring membrane proteins. Both the plant and animal cyt b-561 are involved in transmembrane electron transport using ascorbate as an electron donor. The similarity between these proteins therefore suggests, for the first time, that this transport supports a number of different cell physiological processes. An evolutionary relationship between the plant and animal proteins is presented.     

Mitochondrial ATP Synthase: A Bioinformatic Approach Reveals New Insights About the Roles of Supernumerary Subunits <Emphasis Type="Italic">g</Emphasis> and A6L

The mitochondrial ATP synthase is a membrane protein complex which couples the proton gradient across the mitochondrial inner membrane to the synthesis of ATP from ADP + P_i. The complex is composed of essential subunits for its motor functions and supernumerary subunits, the roles of which remain to be elucidated. Subunits g and A6L are supernumerary subunits, and the specific roles of these subunits are still matters of debate. To gain insight into the functions of these two subunits, we carried out the alignment and the homolog search of the protein sequences of the subunits and found the following features: Subunit g appears to have isoforms in animals, and the transmembrane domain of the animal subunit g contains a completely conserved acidic residue in the middle of a helix on the conserved side of the transmembrane helix. This finding implicates the conserved acidic residue as important for the function of subunit g. The alignment of A6L protein sequences shows a conserved aromatic residue at the N-terminal domain with which the N-terminal MPQL sequence comprises a unique MPQLX₄Ar motif that can signify the protein A6L. The conserved aromatic residue may also be important for the function of A6L.     

Identification of a Novel mRNA-Associated Protein in Oocytes ofPleurodeles waltlandXenopus laevis

Amphibian oocytes accumulate a large pool of mRNA molecules for future embryonic development. Due to their association with specific proteins the stored maternal RNAs are translationally repressed. The identification of these RNA-binding proteins and the characterization of their functional domains may contribute to the understanding of the translational repression mechanisms and the subsequent activation processes during early embryogenesis. Here we present the completePleurodelescDNA sequence of a cytoplasmic protein which is present in oocytes, eggs, and very early cleavage stage embryos but undetectable in postcleavage embryo and adult tissues. The predicted molecular mass of the protein is 55 kDa and the apparent molecular mass as determined by SDS–PAGE, 68 kDa. The deduced amino acid sequence reveals proline- and serine-rich domains in the aminoterminal part as well as two RGG boxes which represent characteristic motifs of several RNA-binding proteins. No distinct homologies to the consensus RNA recognition motif were found. The 55-kDa protein was recovered in cytoplasmic ribonucleoprotein (RNP) particles containing poly(A)⁺RNA. It was therefore termed RAP55 for mRNA-associated protein of 55 kDa. However, a direct interaction of RAP55 with mRNA could not be demonstrated by UV-crosslinking experiments, indicating that it is bound to mRNP complexes via protein–protein interactions. RAP55 is evolutionarily conserved since antibodies raised against a recombinantPleurodelesRAP55 fragment recognize the protein fromPleurodelesandXenopus.The expression pattern and intracellular distribution of RAP55 suggest that it is part of those mRNP particles which are translationally repressed during oogenesis and become activated upon progesterone-induced oocyte maturation.     

Altered patterns ofN-linked glycosylation of theTorpedo acetylcholine receptor expressed inXenopus oocytes

Summary The nicotinic acetylcholine receptor (AChR) fromTorpedo electroplax is an oligomeric transmembrane glycoprotein made up of four highly homologous subunits in a stoichiometry of ₂. The role ofN-linked glycosylation of the AChR has been studied in several cell lines and these studies have suggested that the addition of carbohydrate may be important for receptor expression. WhileXenopus oocytes have proven to be an invaluable tool for studying the AChR, little is known aboutN-linked glycosylation of the oocyte-expressed receptor. The present report demonstrates that the oocyte-expressed AChR is glycosylated and contains the same number of oligosaccharide residues per subunit as the native receptor. However, unlike the nativeTorpedo receptor which contains both high mannose and complex oligosaccharides, the oocyte-expressed AChR contains only high mannose oligosaccharide modifications. However, as has been well documented, theTorpedo AChR expressed in oocytes is fully functional, demonstrating that the precise nature of the oligosaccharide modification is not critical for receptor function.The role of the oligosaccharide component of the AChR in receptor function was examined using tunicamycin (TM) to inhibitN-linked protein glycosylation. TM treatment resulted in a 70–80% inhibition of AChR expression in oocytes. Functional, unglycosylated receptors were not expressed; receptors expressed in TM-treated oocytes were functional wild-type, glycosylated AChR, formed only during the initial 12 hr of TM exposure. These data suggest that while glycosylation of the oocyte-expressedTorpedo AChR is required for assembly of subunits into a functional receptor, as has been demonstrated in other cells, oocyte modification of normalTorpedo glycosylation patterns does not affect receptor function or assembly.     

Functional response of the small multidrug resistance protein EmrE to mutations in transmembrane helix 2

Escherichia coli EmrE is a small multidrug resistance protein encompassing four transmembrane (TM) sequences that oligomerizes to confer resistance to antimicrobials. Here we examined the effects on in vivo protein accumulation and ethidium resistance activity of single residue substitutions at conserved and variable positions in EmrE transmembrane segment 2 (TM2). We found that activity was reduced when conserved residues localized to one TM2 surface were replaced. Our findings suggest that conserved TM2 positions tolerate greater residue diversity than conserved sites in other EmrE TM sequences, potentially reflecting a source of substrate polyspecificity.     

Comparative analysis of the Kekkon molecules,related members of the LIG superfamily

Leucine-rich repeats (LRRs) and immunoglobulin (Ig) domains represent two of the most abundant sequence elements in metazoan proteomes. Despite this prevalence, comparatively few molecules containing both LRR and Ig (LIG) modules exist, and fewer still have been functionally defined. One LIG whose function has been investigated is the Drosophila protein Kekkon1 (Kek1). In vivo studies have demonstrated a role for Kek1 in Epidermal Growth Factor Receptor (EGFR) signaling and have suggested a role in neuronal pathfinding. Kek1 is the founding member of the Kek family, a group of six Drosophila transmembrane proteins that contain seven LRRs and a single Ig in their extracellular domains. While this arrangement of domains predicts a possible role as cell adhesion molecules (CAMs), to date little is known about the function or evolutionary relationship of these additional Kek molecules. Here we report that orthologs of Kek1, Kek2, Kek5, and Kek6 exist in the mosquito, Anopheles gambiae, and the honeybee, Apis mellifera, indicating that this family has been conserved for ~300 million years of evolutionary time. Comparative sequence analyses reveal remarkable identity among these orthologs, primarily in their extracellular regions. In contrast, the intracellular regions are more divergent, exhibiting only small pockets of conservation. In addition, we provide support for the general notion that these molecules may share common functions as CAMs, by demonstrating that Kek family members can form homotypic and heterotypic complexes.Edited by D. TautzChristina M. MacLaren, Timothy A. Evans and Diego Alvarado contributed equally to this work     

Functional involvement of membrane-embedded and conserved acidic residues in the ShaA subunit of the multigene-encoded Na/H antiporter in Bacillus subtilis

ShaA, a member of a multigene-encoded Na⁺/H⁺ antiporter in B. subtilis, is a large integral membrane protein consisting of 20 transmembrane helices (TM). Conservation of ShaA-like protein subunits in several cation-coupled enzymes, including the NuoL (ND5) subunit of the H⁺-translocating complex I, suggests the involvement of ShaA in cation transport. Bacillus subtilis ShaA contains six acidic residues that are conserved in ShaA homologues and are located in putative transmembrane helices. We examined the functional involvement of the six transmembrane acidic residues of ShaA by site-directed mutagenesis. Mutation in glutamate (Glu)-113 in TM-4, Glu-657 in TM-18, aspartate (Asp)-734 and Glu-747 in TM-20 abolished the antiport activity, suggesting that these residues play important roles in the ion transport of Sha. The acidic group was necessary and sufficient in Glu-657 and Asp-743, while it was not true of Glu-113 and Glu-747. Mutation in Asp-103 in TM-3, which is conserved in ShaA-types but not in ShaAB-types, partially affected on the antiport activity. Mutation in Asp-50 in TM-2 resulted in a unexpected phenotype: mutants retained the wild type level of ability to confer NaCl resistance to the Na⁺/H⁺ antiporter-deficient E. coli KNabc, but showed a very low antiport activity. The acidic group of Asp-50 and Asp-103 was not essential for the function. Our results suggested that these acidic residues are functionally involved in the ion transport of Sha, and some of them probably in cation binding and/or translocation.     

<Emphasis Type="Italic">Drosophila</Emphasis> Bys is nuclear and shows dynamic tissue-specific expression during development

Although the bys-like family of genes has been conserved from yeast to humans, it is not apparent to what extent the function of Bys-like proteins has been conserved across phylogenetic groups. Human Bystin is thought to function in a novel cell adhesion complex involved in embryo implantation. The product of the yeast bys-like gene, Enp1, is nuclear and has a role in pre-ribosomal RNA (pre-rRNA) splicing and ribosome biogenesis. To gain insight into the function of the Drosophila melanogaster bys-like family member, termed bys, we examined bys mRNA expression and the localization of Bys protein. In embryos, bys mRNA is expressed in a tissue-specific pattern during gastrulation. In the larval wing imaginal disc, bys mRNA is expressed in the ventral and dorsal regions of the wing pouch, regions that give rise to epithelia that adhere to one another after the wing disc everts. The bys mRNA expression patterns could be interpreted as being consistent with a role for Bys in events requiring cell-cell interactions. However, embryonic bys mRNA expression patterns mirror those of genes that are potential targets of the growth regulator Myc and encode nucleolar proteins implicated in cell growth. Additionally, in Schneider line 2 (S2) cells, an epitope-tagged Bys protein is localized to the nucleus, suggesting that Drosophila Bys function may be conserved with that of yeast Enp1.Edited by D.A. Weisblat     

Vitellogenin motifs conserved in nematodes and vertebrates

Summary Caenorhabditis elegans vitellogenins are encoded by a family of six genes, one of which,vit-5, has been previously sequenced and shown to be surprisingly closely related to the vertebrate vitellogenin genes. Here we report an alignment of the amino acid sequences of vitellogenins from frog and chicken with those from threeC. elegans genes:vit-5 and two newly sequenced genes,vit-2 andvit-6. The four introns ofvit-6 are all in different places from the four introns ofvit-5, but three of these eight positions are identical or close to intron locations in the vertebrate vitellogenin genes. The encoded polypeptides have diverged from one another sufficiently to allow us to draw some conclusions about conserved positions. Many cysteine residues have been conserved, suggesting that vitellogenin structure has been maintained over a long evolutionary distance and is dependent upon disulfide bonds. In addition, a 20-residue segment shows conservation between the vertebrate and the nematode vitellogenins. This sequence may play a highly conserved role in vitellogenesis, such as specific recognition by oocytes. On the whole, however, selection may be acting more strongly on amino acid composition and codon usage than on amino acid sequence, as might be expected for abundant storage proteins: The amino acid compositions ofvit-2, vit-5, andvit-6 products are remarkably similar, despite the fact that the sequence of thevit-2 protein is only 22% and 50% identical to the sequences ofvit-6 andvit-5 proteins, respectively.     

Structure and evolution of opossum,guinea pig,and porcupine cytochrome b genes

Summary We have sequenced the mitochondrial cytochrome b gene from the guinea pig, the African porcupine, and a South American opossum. A phylogenetic analysis, which includes 22 eutherian and four other vertebrate cytochrome b sequences, indicates that the guinea pig and the porcupine constitute a natural clade (Hystricomorpha) that is not a sister group to the clade of mice and rats (Myomorpha). Therefore, the hypothesis that the Rodentia is paraphyletic receives additional support. The artiodactyls, the perissodactyls, and the cetaceans form a group that is separated from the primates and the rodents. The 26 sequences are used to study the structure/function relationships in cytochrome b, whose function is electron transport. Most of the amino acid residues involved in the two reaction centers are well conserved in evolution. The four histidines that are believed to ligate the two hemes are invariant among the 26 sequences, but their nearby residues are not well conserved in evolution. The eight transmembrane domains represent some of the most divergent regions in the cytochrome b sequence. The rate of nonsynonymous substitution is considerably faster in the human and elephant lineages than in other eutherian lineages; the faster rate might be due to coevolution between cytochrome b and cytochrome c. Offprint requests to: W.-H. Li     

Essential roles of snap-29 in C. elegans

SNARE domain proteins are key molecules mediating intracellular fusion events. SNAP25 family proteins are unique target-SNAREs possessing two SNARE domains. Here we report the genetic, molecular, and cell biological characterization of C. elegans SNAP-29. We found that snap-29 is an essential gene required throughout the life-cycle. Depletion of snap-29 by RNAi in adults results in sterility associated with endomitotic oocytes and pre-meiotic maturation of the oocytes. Many of the embryos that are produced are multinucleated, indicating a defect in embryonic cytokinesis. A profound defect in secretion by oocytes and early embryos in animals lacking SNAP-29 appears to be the underlying defect connecting these phenotypes. Further analysis revealed defects in basolateral and apical secretion by intestinal epithelial cells in animals lacking SNAP-29, indicating a broad requirement for this protein in the secretory pathway. A SNAP-29-GFP fusion protein was enriched on recycling endosomes, and loss of SNAP-29 disrupted recycling endosome morphology. Taken together these results suggest a requirement for SNAP-29 in the fusion of post-Golgi vesicles with the recycling endosome for cargo to reach the cell surface.     

Substrate specificity of a chimera made from Xenopus SGLT1-like protein and rabbit SGLT1

To characterize the sugar translocation pathway of Na⁺/glucose cotransporter type 1 (SGLT1), a chimera was made by substituting the extracellular loop between transmembrane domain (TM) 12 and TM13 of Xenopus SGLT1-like protein (xSGLT1L) with the homologous region of rabbit SGLT1. The chimera was expressed in Xenopus oocytes and its transport activity was measured by the two-microelectrode voltage-clamp method. The substrate specificity of the chimera was different from those of xSGLT1L and SGLT1. In addition the chimera's apparent Michaelis-Menten constant (K_m) for myo-inositol, 0.06 mM, was about one fourth of that of xSGLT1L, 0.25 mM, while the chimera's apparent K_m for d-glucose, 0.8 mM, was about one eighth of that of xSGLT1L, 6.3 mM. Our results suggest that the extracellular loop between TM12 and TM13 participates in the sugar transport of SGLT1.     

“HAIRY CANOLA” – Arabidopsis GL3 Induces a Dense Covering of Trichomes on Brassica napus Seedlings

Transformation with the Arabidopsis bHLH gene 35S:GLABRA3 (GL3) produced novel B. napus plants with an extremely dense coverage of trichomes on seedling tissues (stems and young leaves). In contrast, trichomes were strongly induced in seedling stems and moderately induced in leaves of a hairy, purple phenotype transformed with a 2.2 kb allele of the maize anthocyanin regulator LEAF COLOUR (Lc), but only weakly induced by BOOSTER (B-Peru), the maize Lc 2.4 kb allele, or the Arabidopsis trichome MYB gene GLABRA1 (GL1). B. napus plants containing only the GL3 transgene had a greater proportion of trichomes on the adaxial leaf surface, whereas all other plant types had a greater proportion on the abaxial surface. Progeny of crosses between GL3⁺ and GL1⁺ plants resulted in trichome densities intermediate between a single-insertion GL3⁺ plant and a double-insertion GL3⁺ plant. None of the transformations stimulated trichomes on Brassica cotyledons or on non-seedling tissues. A small portion of bHLH gene-induced trichomes had a swollen terminal structure. The results suggest that trichome development in B. napus may be regulated differently from Arabidopsis. They also imply that insertion of GL3 into Brassica species under a tissue-specific promoter has strong potential for developing insect-resistant crop plants. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus

The chalcone synthase is a key enzyme that catalyses the first dedicated reaction of the flavonoid pathway in higher plants. The chs gene and its protein product in rice has been investigated. The presence of a chalcone synthase (CHS) protein in rice seedlings and its developmental stage-specific expression has been demonstrated by western analysis. The chalcone synthase of rice was found to be immunologically similar to that of maize. A rice cDNA clone, Os-chs cDNA, encoding chalcone synthase, isolated from a leaf cDNA library of an indica rice variety Purpleputtu has been mapped to the centromeric region of chromosome 11 of rice. It was mapped between RFLP markers RG2 and RG103. RG2 is the nearest RFLP marker located at a genetic distance of 3.3 cM. Some segments of chromosome 11 of rice including chs locus are conserved on chromosome 4 of maize. The markers, including chs locus on chromosome 11 of rice are located, though not in the same order, on chromosome 4 of maize. Genetic analysis of purple pigmentation in two rice lines, Abhaya and Shyamala, used in the present mapping studies, indicated the involvement of three genes, one of which has been identified as a dominant inhibitor of leaf pigmentation. The Os-chs cDNA shows extensive sequence homology, both for DNA and protein (deduced), to that of maize, barley and also to different monocots and dicots.     

Characterisation of the <Emphasis Type="Italic">Aspergillus niger dapB</Emphasis> gene,which encodes a novel fungal type IV dipeptidyl aminopeptidase

We have cloned the Aspergillus niger dapB gene. Analysis of its nucleotide sequence and the corresponding protein sequence indicates that the gene encodes a type IV dipeptidyl aminopeptidase (DPP IV). Based upon its deduced sequence we predict the presence of a transmembrane domain in the protein. Furthermore, dapB-overexpressing transformants display an increase in intracellular DPP IV activity. This is the first reported characterisation of a dipeptidyl aminopeptidase with a transmembrane domain from a filamentous fungus. Using the dapB sequence as a query, we were able to identify 14 DPP IV-encoding genes, and 12 additional DPPIV proteases in public genomic databases. Phylogenetic analysis reveals that in yeasts there are two clades of genes that encode DPP IV proteases with a transmembrane domain. In this study we demonstrate that, as in yeasts, two classes of DPP IV-encoding genes exist in filamentous fungi. However, only one of these codes for DPP IV proteases with a transmembrane domain. The second type present in filamentous fungi encodes extracellular DPP IV proteases. The dapB gene belongs to the first cluster. We propose that DapB plays a role in the proteolytic maturation of enzymes produced by A. niger.Electronic Supplementary Material Supplementary material is available for this article at     

Ala-504 is a determinant of substrate binding affinity in the mouse Na/dicarboxylate cotransporter

The Na⁺/dicarboxylate cotransporters from mouse (mNaDC1) and rabbit (rbNaDC1) differ in their ability to handle adipate, a six-carbon terminal dicarboxylic acid. The mNaDC1 and rbNaDC1 amino acid sequences are 75% identical. The rbNaDC1 does not transport adipate and only succinate produced inward currents under two-electrode voltage clamp. In contrast, oocytes expressing mNaDC1 had adipate-dependent inward currents that were about 60% of those induced by succinate. In order to identify domains involved in adipate transport, we examined the functional properties of a series of chimeric transporters made between mouse and rabbit NaDC1. We find that multiple transmembrane helices (TM), particularly TM 8, 9, and 10, are involved in adipate transport. In TM 10 there is only one amino acid difference between the two proteins, corresponding to Ala-504 in mouse and Ser-512 in rabbit NaDC1. The mNaDC1-A504S mutant had decreased adipate-dependent currents relative to succinate-dependent currents and an increase in the K_0.5 for both succinate and glutarate. We conclude that multiple amino acids from TM 8, 9 and 10 contribute to the transport of adipate in NaDC1. Furthermore, Ala-504 in TM 10 is an important determinant of K_0.5 for both adipate and succinate.