Isolation of a gene encoding a G protein α subunit involved in the regulation of cAMP levels in the yeast Kluyveromyces lactis

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0·5 kb (P-1) and 0·8 kb (P-2). Sequencing showed that these two fragments share high homology with genes coding for the Gα subunits from different sources. Using the P-1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein α subunits from Saccharomyces cerevisiae. KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this Gα subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian Gαs family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence. The sequence reported in this paper has been deposited in the GenBank data base (Accession No. L45105).     

Cloning and characterization of WMSU1, a Williopsis saturnus var. mrakii gene encoding a new yeast SUN protein involved in the cell wall structure

SUN proteins of Saccharomyces cerevisiae have been defined on the basis of high homologies in their C-terminal domain. Recently, two of these four proteins were shown to be involved in cell wall morphogenesis (Mouassite et al., 2000a). In the present study, we have isolated WMSU1 (Accession No. AF418983), a new SUN-related gene, from W. saturnus var. mrakii MUCL 41968. Sequencing of the gene revealed an open reading frame coding for 402 amino acids. The predicted amino acid sequence of WMSU1 is closely related to the S. cerevisiae SUN proteins and to other yeast proteins involved in cell wall metabolism. WMSU1 is proposed to encode a cell wall protein since its predicted product contains a signal sequence, a Kex2p cleavage site and a serine/threonine-rich N-terminal domain. Southern blot analysis of the W. saturnus var. mrakii MUCL 41968 genome using the highly conserved domain of WMSU1 as a probe suggested that the isolated gene belongs to a multigenic family. Expression of WMSU1 in E. coli led to a 45 kDa protein, which appeared to be toxic to this host. Scanning electron microscopy analysis of a recombinant S. cerevisiae producing Wmsu1p showed that this strain exhibited an altered cell wall, thus pointing to a probable role of this protein in the cell wall structure.     

Production of a high‐protein meal and fermentable sugars from defatted soybean meal,a co‐product of the soybean oil industry

The goal of this research was to produce a high‐protein meal by treating defatted soybean meal, a by‐product of soybean oil production, with dilute acid. Treatments were a mild hydrolysis at 80 °C with sulphuric acid at concentrations ranging from 0.5% to 2.0% (w/v) and times varying from 1 to 16 h that were arranged according to a central composite rotatable experimental design. The end products were an enhanced‐protein meal and a carbohydrate concentrate of fermentable and nonfermentable sugars. The highest protein content rise, from 48% to 58%, was for treatments with concentrations of acid ranging between 1.2% and 1.7% and times between 1.0 and 2.6 h. The maximum yield of fermentable sugar was 21.0% d.b. at 2.0% H₂SO₄ and treatments of at least 6 h. The conditions that provide a highest protein and sugar contents were the treatments with concentrations of sulphuric acid ranging from 0.9 to 1.9% H₂SO₄ for 1–4 h.     

Isolation of conglutin δ, a sulphur-rich protein from the seeds of Lupinus angustifolius L.

Although a 2S globulin class has been observed in salt extracts from seeds of several lupin species, there have been conflicting reports regarding the importance of this class in Lupinus angustifolius. Conglutin δ, a major sulphur-rich protein extracted from mature seeds of L. angustifolius cv. Uniwhite, was separated by gel-filtration into two oligomeric forms. The sedimentation coefficients of conglutin δ₁ (20%) and conglutin δ₂ (80%), were 3.2S and 2.0S respectively. The amino acid compositions of both oligomeric forms of conglutin δ were identical and similar to the compositions published for the 2S globulins in other lupin species. Because of the low level of tyrosine and the absence of tryptophan, conglutins δ₁ and δ₂ showed little absorbance at 280nm (E^1%/1 cm^?23). They were characterised by unusually high levels of glutamic acid and 1/2 cysteine (38.5 and 8.5 residues percent respectively) while methionine was absent. Gradient SDS-PAGE showed that conglutins δ₁ and δ₂ were homogeneous single-subunit species. On reduction, with or without S-carboxymethylation, both the conglutin δ₁ and δ₂ subunits yielded similar pairs of large and small polypeptide chains. Since conglutin δ rarely resolves from conglutin a during electrophoresis on cellulose acetate membranes in phosphate buffer at neutral pH, this method is not as useful for screening lupin cultivars as has been claimed previously.     

Biochemical and genetic characterization of Hmi1p, a yeast DNA helicase involved in the maintenance of mitochondrial DNA

The HMI1 gene encodes a DNA helicase that localizes to the mitochondria and is required for maintenance of the mitochondrial DNA (mtDNA) genome of Saccharomyces cerevisiae. Identified based on its homology with E. coli uvrD, the HMI1 gene product, Hmi1p, has been presumed to be involved in the replication of the 80 kb linear S. cerevisiae mtDNA genome. Here we report the purification of Hmi1p to apparent homogeneity and provide a characterization of the helicase reaction and the ATPase reaction with regard to NTP preference, divalent cation preference and the stimulatory effects of different nucleic acids on Hmi1p-catalysed ATPase activity. Genetic complementation assays indicate that mitochondrial localization of Hmi1p is essential for its role in mtDNA metabolism. The helicase activity, however, is not essential. Point mutants that lack ATPase/helicase activity partially complement a strain lacking Hmi1p. We suggest several possible roles for Hmi1p in mtDNA metabolism.     

The role of PaAAC1 encoding a mitochondrial ADP/ATP carrier in the biosynthesis of extracellular glycolipids,mannosylerythritol lipids,in the basidiomycetous yeast Pseudozyma antarctica

Pseudozyma antarctica produces large amounts of the glycolipid biosurfactants known as mannosylerythritol lipids (MEL), which show not only excellent surface‐active properties but also versatile biochemical actions. A gene homologous with a mitochondrial ADP/ATP carrier was dominantly expressed in P. antarctica under MEL‐producing conditions on the basis of previous gene expression analysis. The gene encoding the mitochondrial ADP/ATP carrier of P. antarctica (PaAAC1) contained a putative open reading frame of 954 bp and encodes a polypeptide of 317 amino acids. The deduced translation product shared high identity of 66%, 70%, 69%, 74%, 75% and 52% with the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae (AAC1), S. cerevisiae (AAC2), S. cerevisiae (AAC3), Kluyveromyces lactis (KlAAC), Neurospora crassa (NcAAC) and human (ANT1), respectively, and conserved the consensus sequences of all ADP/ATP carrier proteins. The gene expression by introducing a plasmid pUXV1‐PaAAC1 into the yeast cells increased the MEL production. In addition, the expression of PaAAC1 in which the conserved arginine and leucine required for ATP transport activity were replaced with isoleucine and serine, respectively, failed to increase MEL production. Accordingly, these results suggest that PaAAC1 encoding a mitochondrial ADP/ATP carrier should be involved in MEL biosynthesis in the yeast. Copyright © 2010 John Wiley & Sons, Ltd.     

SpOPT1, a member of the oligopeptide family (OPT) of the fission yeast Schizosaccharomyces pombe,is involved in the transport of glutathione through the outer membrane of the cell

A protein involved in the transport of glutathione has been identified, cloned and characterized from the fission yeast Schizosaccharomyces pombe. Database searches revealed the Sz. pombe ORF SPAC29B12.10c as a close homologue to several members of the OPT family, including the Saccharomyces cerevisiae high‐affinity glutathione transporter Hgt1p. The gene product of SPAC29B12.10c has been identified as a protein, named SpOPT1, localized within the plasma membrane, transporting the tripeptide glutathione. Disruption of SPAC29B12.10c led to strains inable to grow on media containing glutathione as a sole source of sulphur, due to the inability to internalize the tripeptide. Disruptants contained significantly less glutathione than wild‐type cells. Furthermore, ΔSpopt1 strains were non‐viable in a glutathione biosynthesis‐defective (Δgsh2) background. However, it was possible to complement the disruption of Spopt1 by overexpressing the intact ORF in the disrupted strain. Copyright © 2009 John Wiley & Sons, Ltd.     

Immunological one‐step determination of the central nervous system indicator proteins,neuron‐specific enolase and glial fibrillary acidic protein,in meat products

The determination of specific marker proteins is important in the prevention of infections and transmission of disease. Several diagnostic assays have been developed but these are mostly restricted to the detection of single antigens. Thus there is a need for multiplex detection assays for the simultaneous analysis of several disease indicators. Consumer protection against the transmission of prion diseases is ensured by the removal of specified risk material from cattle meat during slaughtering and this is regulated by law. The investigation reported here describes a one‐step determination on immunoblots of the simultaneous detection of two indicator proteins, neuron‐specific enolase and glial fibrillary acidic protein, in tissues of the central nervous system. Although the two proteins run in polyacrylamide gels with similar molecular masses the indicators are differentiated by immunological reactions followed by visualizing the different coloured specific protein bands which develop. The enolase exhibits a brown band, whereas the glial fibrillary acidic protein is red. Immunoblotting has proved to be a suitable assay for multiplex analysis of marker proteins possessing similar molecular weights and is therefore a suitable tool for application in food, veterinary and medical facilities.     

The subunit structure and stability of conglutin δ, a sulphurrich protein from the seeds of Lupinus angustifolius L.

The oligomeric structure and stability of conglutin δ, the 2S sulphur-rich lupin seed protein, has been studied. Molecular weights were determined by sedimentation equilibrium methods in the analytical ultracentrifuge. Conglutin δ₂ (M_r 14 000, 2S), the most abundant form, was composed of one large (?9600) and one small polypeptide chain linked by disulphide bonds. The minor oligomeric form, conglutin δ₁ (M_r 28 000, 2.8S), was a disulphide-linked dimer of conglutin δ₂. Each form was capable of reversible association and at low ionic strength (neutral pH), the conglutin δ₁ momomer associated to a homogeneous dimer (M_r 56 000, 4.1S). Calculated frictional ratios (f/f₀ ? 1.28–1.49) suggested that the three different forms were asymmetric. Spectral studies (ORD/CD) showed that conglutins δ₁ and δ₂ were rich in alpha-helix (?38%) unlike the major 7S and 11S legume seed storage proteins. The helical structure was unusually stable both to heat and chemical denaturants; below 60°C (at neutral pH) the helix remained unaffected and only partial denaturation occurred at higher temperatures. Nevertheless, complete denaturation was achieved (at 20°C) in high concentrations of guanidine hydrochloride (greater than 6.5 M). The stability was due to the presence of disulphide cross-links; with the addition of reducing agent, as little as 1 M guanidine hydrochloride (GuHCl) was sufficient for denaturation of the helical structure. Titration experiments showed a single buried tyrosine (pK 11.4) which could be exposed (pK 10.2) in 6 M GuHCl.     

Effect of autochthonous starter cultures in the production of “salchichón”, a traditional Iberian dry-fermented sausage, with different ripening processes

The aim of the study was to determine how two different ripening processes affected the development of lactic acid bacteria (LAB) plus Staphylococcus autochthonous starter cultures, and the physico-chemical and sensory characteristics of the Iberian dry-fermented sausages (Salchichón). Each of two strains of Pediococcus acidilactici (MS198 and MS200) and one of Staphylococcus vitulus (RS34) were associated to prepare two starter cultures: P198S34 and P200S34. Then salchichón was prepared following two different manufacturing procedures. Both starter cultures were able to compete well and colonize the sausages, although P200S34 was better adapted to process 1 ripening conditions. There were evident differences shown by the texture analysis, with the control batches being tougher. Also, the highest biogenic amine levels were found in control batches. While the use of these starter cultures does not produce a negative effect on the sensory characteristics of these traditional fermented sausages, it can improve their homogeneity and healthiness but an appropriate combination of strains should be chosen for each process.     

Assembly of alcohol oxidase in the cytosol of a peroxisome-deficient mutant of Hansenula polymorpha—properties of the protein and architecture of the crystals

We have studied the expression of alcohol oxidase (AO) in a peroxisome-deficient mutant strain of Hansenula polymorpha. High levels of octameric, active AO (up to 3·0 U/mg protein) were detected in cells grown at low dilution rates in a glucose-limited chemostat in the presence of choline as the sole nitrogen source. Monomeric or other intermediate forms of AO were not detected in the mutant strain. This indicated that assembly of the protein into active octameric molecules in the cytosol was as efficient as in wild-type cells where this process is confined to the peroxisomal matrix. At relatively low rates of expression (less than 1 U/mg protein) AO was localized throughout the cytosol and, surprisingly, was also present inside the nucleus. However, at enhanced levels large crystalloids were formed. Generally one crystalloid was observed per cell, whereas smaller ones were occasionally found in developing buds. Also large crystalloids have been observed inside the nucleus. These crystalloids were not surrounded by a membrane. Based on the morphology of the molecules that constituted these crystalloids and the results of (immuno)cytochemical experiments we conclude that the crystalloids are composed of octameric AO molecules, arranged in a regular lattice, identical to the 3-dimensional architecture previously described for the crystalline matrix of peroxisomes in methanol-grown wild type cells of H. polymorpha. Attempts to purify the crystalloids by conventional fractionation methods failed, due to their apparent fragility; however, (immuno)cytochemical experiments revealed that catalase and dihydroxyacetone synthase were also associated with these structures.