Isolation from Candida albicans of a functional homolog of the Saccharomyces cerevisiae KRE1 gene, which is involved in cell wall beta-glucan synthesis.

The KRE1 gene of Saccharomyces cerevisiae, sacKRE1, appears to be involved in the synthesis of cell wall beta-glucan. S. cerevisiae strains with mutations in the KRE1 gene produce a structurally altered cell wall (1----6)-beta-glucan, which results in resistance to K1 killer toxin. We isolated the canKRE1 gene from Candida albicans by its ability to complement a kre1 mutation in S. cerevisiae and confer sensitivity to killer toxin. Sequence analysis revealed that the predicted protein encoded by canKRE1 shares an overall structural similarity with that encoded by sacKRE1. The canKRE1 protein is composed of an N-terminal signal sequence, a central domain of 46% identity with the sacKRE1 protein, and a C-terminal hydrophobic tract. These structural and functional similarities imply that the canKRE1 gene carries out a function in C. albicans cell wall assembly similar to that observed for sacKRE1 in S. cerevisiae.     

Isolation of Penicillium chrysogenum PEX1 and PEX6 encoding AAA proteins involved in peroxisome biogenesis

In Penicillium chrysogenum, key enzymes involved in the production of penicillin reside in peroxisomes. As a first step to understand the role of these organelles in penicillin biosynthesis, we set out to isolate the genes involved in peroxisome biogenesis. Here we report the cloning and characterization of P. chrysogenum PEX1 and PEX6, which encode proteins of the AAA family of ATPases. The second AAA module, which is essential for the function of Pex1p and Pex6p in peroxisome biogenesis, is highly conserved in both PcPex1p and PcPex6p. PcPEX1 and PcPEX6 contain three and two introns, respectively. Received: 15 February 2000 / Accepted: 18 February 2000     

Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins.

The alternative, heterometal-free nitrogenase of Rhodobacter capsulatus is repressed by traces of molybdenum in the medium. Strains carrying mutations located downstream of nifB copy II were able to express the alternative nitrogenase even in the presence of high molybdate concentrations. DNA sequence analysis of a 5.5-kb fragment of this region revealed six open reading frames, designated modABCD, mopA, and mopB. The gene products of modB and modC are homologous to ChlJ and ChlD of Escherichia coli and represent an integral membrane protein and an ATP-binding protein typical of high-affinity transport systems, respectively. ModA and ModD exhibited no homology to known proteins, but a leader peptide characteristic of proteins cleaved during export to the periplasm is present in ModA, indicating that ModA might be a periplasmic molybdate-binding protein. The MopA and MopB proteins showed a high degree of amino acid sequence homology to each other. Both proteins contained a tandem repeat of a domain encompassing 70 amino acid residues, which had significant sequence similarity to low-molecular-weight molybdenum-pterin-binding proteins from Clostridium pasteurianum. Compared with that for the parental nifHDK deletion strain, the molybdenum concentrations necessary to repress the alternative nitrogenase were increased 4-fold in a modD mutant and 500-fold in modA, modB, and modC mutants. No significant inhibition of the heterometal-free nitrogenase by molybdate was observed for mopA mopB double mutants. The uptake of molybdenum by mod and mop mutants was estimated by measuring the activity of the conventional molybdenum-containing nitrogenase. Molybdenum transport was not affected in a mopA mopB double mutant, whereas strains carrying lesions in the binding-protein-dependent transport system were impaired in molybdenum uptake.     

Identification of a multigene family encoding putative beta-glucan-binding proteins in Medicago truncatula

Branched 1,6-1,3-beta-glucans from Phytophthora sojae cell walls represent pathogen-associated molecular patterns (PAMPs) that have been shown to mediate the activation of plant defence reactions in many legumes. In soybean, a receptor protein complex containing a high affinity beta-glucan-binding protein (GBP) was identified and investigated in detail. In the model legume Medicago truncatula, used for functional genomic studies of various plant-microbe interactions, a high-affinity beta-glucan-binding site was characterized biochemically. However, to date, none of the genes encoding GBPs from M. truncatula have been described. Here, we report the identification of four full-length clones encoding putative beta-glucan-binding proteins from M. truncatula, MtGBP1, 2, 3, and 4, composing a multigene family encoding GBP-related proteins in this plant. Differences in expression patterns as well as in regulation on treatment with two different biotic elicitors are demonstrated for the members of the GBP family and for a selection of defence-related genes.     

Structure and function of fas-1A, a gene encoding a putative fatty acid synthetase directly involved in aflatoxin biosynthesis in Aspergillus parasiticus.

A novel gene, fas-1A, directly involved in aflatoxin B1 (AFB1) biosynthesis, was cloned by genetic complementation of an Aspergillus parasiticus mutant strain, UVM8, blocked at two unique sites in the AFB1 biosynthetic pathway. Metabolite conversion studies localized the two genetic blocks to early steps in the AFB1 pathway (nor-1 and fas-1A) and confirmed that fas-1A is blocked prior to nor-1. Transformation of UVM8 with cosmids NorA and NorB restored function in nor-1 and fas-1A, resulting in synthesis of AFB1. An 8-kb SacI subclone of cosmid NorA complemented fas-1A only, resulting in accumulation of norsolorinic acid. Gene disruption of the fas-1A locus blocked norsolorinic acid accumulation in A. parasiticus B62 (nor-1), which normally accumulates this intermediate. These data confirmed that fas-1A is directly involved in AFB1 synthesis. The predicted amino acid sequence of fas-1A showed a high level of identity with extensive regions in the enoyl reductase and malonyl/palmityl transferase functional domains in the beta subunit of yeast fatty acid synthetase. Together, these data suggest that fas-1A encodes a novel fatty acid synthetase which synthesizes part of the polyketide backbone of AFB1. Additional data support an interaction between AFB1 synthesis and sclerotium development.     

Selection of cDNAs encoding putative type II membrane proteins on the cell surface from a human full-length cDNA bank

We have developed a simple method to test whether a hydrophobic segment near the N-terminus of a protein functions as a type II signal anchor (SA) in which the N-terminus faces the cytoplasm. A cDNA fragment containing the putative SA sequence of a target clone was fused in-frame to the 5' end of a cDNA fragment encoding the protease domain of urokinase-type plasminogen activator (u-PA). The resulting fused gene was expressed in COS7 cells. Fibrinolytic activity on the cell surface was measured by placing a fibrin sheet in contact with the transfected COS7 cells after removing the medium. When the cDNA fragment encoded a SA, the fibrin sheet was lysed by the u-PA expressed on the cell surface. The fibrinolytic activity was not detected in the culture medium, suggesting that the u-PA remains on the cell surface anchored via the SA in the membrane without being cleaved by signal peptidase. This fibrin sheet method was successfully applied to select five novel cDNA clones encoding putative type II membrane proteins from a human full-length cDNA bank.     

Forces involved in the assembly and stabilization of membrane proteins.

Hydrophobic organization: Determination of the structure of the bacterial photosynthetic reaction center, bacterial porins, and bacteriorhodopsin allows a comparison of the basic structural features of integral membrane proteins. Structure parameters of membrane- and water-soluble proteins are surprisingly similar, given the different dielectric environments, except for the polarity of residues on the protein surface. Hydrophobic and electrostatic forces: 1) Intramembrane helix-helix interactions that are sensitive to small structure changes can dictate assembly of membrane proteins, as indicated by reconstitution of bacteriorhodopsin from proteolytic fragments and specific dimer formation of the human erythrocyte sialoglycoprotein glycophorin A. 2) Electrostatic interactions have an important role in determining the trans-membrane orientation of integral membrane proteins of the bacterial inner membrane, as expressed by the "positive-inside" rule for the distribution of basic residues on the cis relative to the trans side of the membrane-spanning alpha-helices. The use of this charge asymmetry rule, in conjunction with a hydrophobicity algorithm for prediction of membrane-spanning domains, allows accurate prediction of the folding patterns of such polypeptides across the membrane. A role of electrostatic interactions in assembly and maintenance of the structure of oligomeric integral membrane protein complexes is also implied by the separation and extrusion from the membrane, at high pH, of the major hydrophobic subunits of the cytochrome b6f complex from the chloroplast thylakoid membrane. It is inferred that the hydrophobic helix-helix interactions between the subunits of this complex, whose function is electron transfer and proton translocation, are relatively weak compared to those in bacteriorhodopsin.     

Genetic diversity and genomic distribution of homologs encoding NBS-LRR disease resistance proteins in sunflower

Three-fourths of the recognition-dependent disease resistance genes (R-genes) identified in plants encode nucleotide binding site (NBS) leucine-rich repeat (LRR) proteins. NBS-LRR homologs have only been isolated on a limited scale from sunflower (Helianthus annuus L.), and most of the previously identified homologs are members of two large NBS-LRR clusters harboring downy mildew R-genes. We mined the sunflower EST database and used comparative genomics approaches to develop a deeper understanding of the diversity and distribution of NBS-LRR homologs in the sunflower genome. Collectively, 630 NBS-LRR homologs were identified, 88 by mining a database of 284,241 sunflower ESTs and 542 by sequencing 1,248 genomic DNA amplicons isolated from common and wild sunflower species. DNA markers were developed from 196 unique NBS-LRR sequences and facilitated genetic mapping of 167 NBS-LRR loci. The latter were distributed throughout the sunflower genome in 44 clusters or singletons. Wild species ESTs were a particularly rich source of novel NBS-LRR homologs, many of which were tightly linked to previously mapped downy mildew, rust, and broomrape R-genes. The DNA sequence and mapping resources described here should facilitate the discovery and isolation of recognition-dependent R-genes guarding sunflower from a broad spectrum of economically important diseases.     

Cloning and characterization of CSP37, a novel gene encoding a putative membrane protein of Candida albicans.

In the course of an analysis of the functions and assembly of the cell wall of Candida albicans, we have cloned and characterized a gene, which we designated CSP37 (cell surface protein), encoding a 37-kDa polypeptide which is a membrane-associated protein. The gene was isolated by immunological screening of a DNA library constructed from mycelial cells with a polyclonal serum raised against cell walls of this morphology. Analysis of the nucleotide sequence of a corresponding genomic DNA fragment revealed a single open reading frame which encodes a predicted protein of 321 amino acids with no significant homology to others in the databases. Disruption of the CSP37 gene by the method described by Fonzi and Irwin (Genetics 134:717-728, 1993) eliminated expression of the Csp37 protein. The mutant strains showed no apparent defect in cell viability, growth, or cell wall assembly but displayed attenuated virulence in systemic infections induced in mice and reduced the ability to adhere to polystyrene.     

Developmental and regional expression and localization of mRNAs encoding proteins involved in RNA translocation.

RNA localization is a regulated component of gene expression of fundamental importance in development and differentiation. Several RNA binding proteins involved in RNA localization during development in Drosophila have been identified, of which Y14, Mago, Pumilio, and IMP-1 are known to be expressed in adult mammalian intestine. The present study was undertaken to define the developmental and regional expression of these proteins, as well as Staufen-1, in mouse intestinal cells and in other tissues and cell lines using RT-PCR, and localization using in situ hybridization and immunohistochemistry. Staufen-1, Y14, Mago-m, and Pumilio-1 were expressed in intestinal epithelial cells of both villus and crypt and in Caco-2 and IEC-6 cells. In contrast, expression of IMP-1 was age- and region-specific, showing clear expression in distal fetal and newborn intestine, but very low or no expression in adult. The mRNAs were cytosolic, with more apical than basal expression in enterocytes. Staufen protein showed a similar localization pattern to that of its cognate mRNA. Overall, the data suggest an essential role for these proteins in intestinal cells. Age and regional expression of IMP-1 may indicate a role in regulation of site-specific translation of intestinal genes or in RNA localization.     

The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins.

60% of the peripheral membrane skeleton of Euglena gracilis consists of equimolar amounts of two proteins (articulins) with M(r)s in SDS gels of 80 and 86 kD. To understand eventually how these proteins assemble and function in maintaining cell form and membrane integrity we have undertaken a molecular characterization of articulins. A lambda gt11 expression library constructed from Euglena gracilis mRNAs was screened with antibodies against both articulins. Two sets of cDNAs were recovered, and evidence from three independent assays confirmed that both sets encoded articulins: (a) Anti-articulin antibodies recognized a high molecular weight beta-galactosidase (beta-gal) fusion protein expressed in bacteria infected with lambda gt11 cDNA clones. (b) Antibodies generated against the bacterially expressed beta-gal fusion protein identified one or the other articulin in Western blots of Euglena proteins. These antibodies also localized to the membrane skeletal region in thin sections of Euglena. (c) Peptide maps of the beta-gal fusion protein were similar to peptide maps of Euglena articulins. From the nucleotide sequence of the two sets of cDNAs an open reading frame for each articulin was deduced. In addition to 37% amino acid identity and overall structural similarity, both articulins exhibited a long core domain consisting of over 30 12-amino acid repeats with the consensus VPVPV--V--. Homology plots comparing the same or different articulins revealed larger, less regular repeats in the core domain that coincided with predicted turns in extended beta-sheets. Outside the core domain a short hydrophobic region containing four seven-amino acid repeats (consensus: APVTYGA) was identified near the carboxy terminus of the 80-kD articulin, but near the amino terminus of the 86-kD articulin. No extensive sequence similarities were found between articulins and other protein sequences in various databanks. We conclude that the two articulins are related members of a new class of membrane cytoskeletal proteins.     

The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1----6)-beta-D-glucan synthesis and normal cell growth.

Yeast kre mutants define a pathway of cell wall (1----6)-beta-D-glucan synthesis, and mutants in genes KRE5 and KRE6 appear to interact early in such a pathway. We have cloned KRE5, and the sequence predicts the product to be a large, hydrophilic, secretory glycoprotein which contains the COOH-terminal endoplasmic reticulum retention signal, HDEL. Deletion of the KRE5 gene resulted in cells with aberrant morphology and extremely compromised growth. Suppressors to the KRE5 deletions arose at a frequency of 1 in 10(7) to 1 in 10(8) and permitted an analysis of deletions which were found to contain no alkali-insoluble (1----6)-beta-D-glucan. These results indicate a role for (1----6)-beta-D-glucan in normal cell growth and suggest a model for sequential assembly of (1----6)-beta-D-glucan in the yeast secretory pathway.