MAL63 codes for a positive regulator of maltose fermentation in Saccharomyces cerevisiae

Summary Genetic analysis of the MAL6 locus has previously yielded mal6 mutants which fall into a single complementation group and which are noninducible for maltase and maltose permease. However, the strains used in these studies contained additional partially functional copies of MAL1 (referred to as MAL1g) and MAL3 (referred to as MAL3g). Using a strain lacking MALg genes, we have isolated two classes of mutants and these classes correspond to mutations in MAL63 and MAL61, two genes of the MAL6 complex. Disruptions of MAL63 are noninducible for maltase and maltose permease and for their corresponding mRNAs. The mal6 mutants are shown to map to MAL63 Inducer exclusion as a cause of the noninducible phenotype of the mal63 mutations has been eliminated by constructing a ma163 mutant in a strain constitutive for maltose permease; the strain remains noninducible. These results rigorously demonstrate that MAL63 is a regulatory gene which plays a positive role in the regulation of maltose fermentation.     

Sequences in the N-terminal cytoplasmic domain of Saccharomyces cerevisiae maltose permease are required for vacuolar degradation but not glucose-induced internalization

In Saccharomyces cerevisiae, glucose addition to maltose fermenting cells causes a rapid loss of maltose transport activity and ubiquitin-mediated vacuolar proteolysis of maltose permease. GFP-tagged Mal61 maltose permease was used to explore the role of the N-terminal cytoplasmic domain in glucose-induced inactivation. In maltose-grown cells, Mal61/HA-GFP localizes to the cell surface and, surprisingly, to the vacuole. Studies of end3Δ and doa4Δ mutants indicate that a slow constitutive internalization of Mal61/HA-GFP is required for its vacuolar localization. Site-specific mutagenesis of multiple serine/threonine residues in a putative PEST sequence of the N-terminal cytoplasmic domain of maltose permease blocks glucose-induced Mal61p degradation but does not affect the rapid loss of maltose transport activity associated with glucose-induced internalization. The internalized multiple Ser/Thr mutant protein co-localizes with Snf7p in a putative late endosome or E-compartment. Further, alteration of a putative dileucine [D/EExxxLL/I] motif at residues 64–70 causes a significant defect in maltose transport activity and mislocalization to an E-compartment but appears to have little impact on glucose-induced internalization. We conclude that the N-terminal cytoplasmic domain of maltose permease is not the target of the signaling pathways leading to glucose-induced internalization of Mal61 permease but is required for its subsequent delivery to the vacuole for degradation.     

Trehalose and maltose metabolism in yeast transformed by a MAL4 regulatory gene cloned from a constitutive donor strain

Summary A 6.8 kb fragment of DNA containing the regulatory sequence MAL4p has been cloned from a genomic library prepared from Saccharomyces cerevisiae strain 1403-7A which ferments maltose constitutively. The library was prepared by ligation of 5–20 kb Sau3AI restriction fragments of total yeast DNA into the BamH1 restriction site of shuttle vector YEp13. A restriction map of the cloned fragment indicates that it encompasses a 2.6 kb segment which closely resembles the regulatory MAL6 gene previously identified (Needleman et al. 1984). The hybrid plasmid, p(MAL4p)4, could transform maltose-nonfermenting strains which contain cryptic -glucosidase and maltose permease genes (malp MALg), but could not transform strains containing a functional regulatory sequence and a defective maltase-permease region (MAlp malg). A correlated absence of maltase and permease DNA from the cloned fragment was indicated by the restriction map. Although the cloned DNA fragment was derived from a constitutive strain, maltose fermentation and -glucosidase formation by yeast transformed with p(MAL4p)4 was largely inducible by maltose and sensitive to catabolite repression. Moreover, the active trehalose accumulation pattern (TAC(+) phenotype) linked to the complete MAL4 locus in strain 1403-7A and other constitutive MAL strains (Oliveira et al. 1981b) was not found in p(MAL4p)4 transformants. It may be concluded that constitutivity of maltose fermentation and the associated active trehalose accumulation are not merely consequences of a cis-dominant mutation causing constitutive formation of the MALp regulatory product. Moreover, constitutivity may not be caused solely by a mutation within the structural region of the MALp gene.     

The MAL63 gene of Saccharomyces encodes a cysteine-zinc finger protein

Summary Inducible maltose fermentation by tSaccharomyces carlesbergensis requires the product of the MAL63 gene of the MAL6 locus. It has been suggested that this gene product is an activator protein controlling the expression of the structural genes encoding the maltose fermentative enzymes perhaps by binding to DNA sequences upstream of these genes. We report the sequence of the MAL63 gene. A single open reading frame is seen capable of encoding a protein of 470 amino acid residues. The deduced sequence of this protein indicates that it is a cysteine-zinc forger protein supporting the hypothesis that the MAL63 gene product is a DNA binding protein.     

Mapping of novel autoreactive epitopes of the diabetes-associated autoantigen IA-2

IA-2, a member of the tyrosine phosphatase family, has been identified as a dominant autoantigen in type 1 diabetes. To define humoral IA-2 epitopes, we generated a panel of IA-2 deletion mutants and chimeric proteins using the highly homologous tyrosine phosphatase-like protein IA-2beta. Analysis of autoantibody reactivity in 111 IA-2 antibody positive sera from patients with type 1 diabetes revealed that humoral epitopes cluster to several domains of the intracytoplasmic part of IA-2 [IA-2ic, amino acid (aa) 604-979]. Immunodominant epitopes were found in the first N-terminal 73 amino acids (56% positive), in the middle domain residing between residues 699-874 (45% positive) and the C-terminus depending on the presence of aa 931-979 (at least 37% positive). Competition experiments with overlapping peptides revealed that autoantibody binding towards the N-terminus was dependent on residues 621-628. In the C-terminal domain, two novel conformation-dependent epitopes were identified. The first epitope requires the presence of the C-terminal part of IA-2 (aa 933-979) and an IA-2-specific region between residues 771-932. Reactivity against the second epitope was dependent on intact C-terminal domains as well as residues in the middle (aa 887-932) and N-terminal regions (aa 604-771) which are conserved in IA-2 and IA-2beta. We here defined novel autoantigenic determinants in the N-terminus of IA-2 and characterized conformational epitopes residing in the C-terminal region or spanning from C-terminal residues to the N-terminal domain of IA-2ic. The identification of dominant target regions of diabetes-specific autoantibodies may help to elucidate the molecular mechanisms involved in the autoimmunity towards IA-2.     

Insertion of non-homologous DNA sequences into a regulatory gene cause a constitutive maltase synthesis in yeast

Summary Two maltase constitutive alleles MAL1-1 ^c and MAL1-2 ^c were obtained as revertants from a defective mall-1 mutant allele not promoting maltose fermentation. Classical genetical analysis showed that the mutations were linked or allelic to the MAL1 locus. Dominance relations were established by testing -glucosidase activities in diploids containing various allele combinations.The maltose regulatory genes belonging to the MAL1, MAL1-1 ^c and MAL1-2 ^c alleles were cloned. Differences in restriction sites were found between the wild type MAL1 and the derived MAL1-constitutive alleles. The MAL1 regulatory gene was located in a 1.15 kb EcoRI fragment (Rodicio and Zimmermann 1985a, b). An EcoRI fragment of this size was found in plasmids containing the MAL1 regulatory wild type allele but was absent from plasmids carrying the constitutive alleles.The genomic organization of the MAL loci in the constitutive mutants was confirmed by Southern analysis. Various fragments containing sequences of the different MAL1 alleles were used to probe genomic digests of MAL1, MAL1-1 ^c and MAL1-2 ^c strains. The results obtained support the conclusion that the constitutive mutations had arisen by a rearrangement between the original mal1-1 mutant allele and sequences with different location in the genome.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Allergenic and antigenic determinants of latex allergen Hev b 1: peptide mapping of epitopes recognized by human, murine and rabbit antibodies

Background The rubber elongation factor in Hevea rubber (Hev b 1) is one of the most important latex allergen and is leading cause oflatex type 1 hypersensitivity in children with spina bifida. Objective The aim of this study was to define the allergenic and antigenic epitopes of Hev b 1. Methods The immunoglobulin- (Ig)E and IgG antibody binding sites on Hev b 1 allergen were delineated by enzyme linked immunosorbent assay (ELISA) using synthetic overlapping peptides covering the whole Hev b 1 sequence. In order to improve the binding capacity and specificity all peptides were biotinylated at the N-terminal end via a 6-aminohexanoic acid as spacer and then adsorbed to streptavidin pre-coated microtitre plates. Fine mapping to define the essential amino acid residues for the antibody binding was achieved by using overlapping peptides with one amino acid offset. Results It was demonstrated that the IgE epitopes were located in different regions of Hev b 1 including the C-terminal segment (121–137) and the segments with amino acid residues of 30–49 and 46–64. Two monoclonal antibodies (MoAbs) II2F3 and II4G9 raised against purified Hev b 1 recognized the C-terminal segment only. The results of epitope mapping with three rabbit antisera revealed that five positive peptides, including the epitope peptides 31–49, 46–64 and 121–137, were involved in the antibody-binding sites. Eine mapping on the segments 46–64 and 121–137 showed that the two MoAbs reacted with the peptide 125–134 in the C-terminal region, whereas the peptide with amino acids 124–134 was essential for recognition by human IgE antibodies. Epitopes to rabbit polyclonal IgG and human IgE were also found to be involved in the amino acid residues of 47–59. Conclusion Our results indicate that the most allergenic/antigenic portions of Hev b 1 allergen are the C-terminal region and the region with amino acid residues of 31–64. In both regions, the minimal IgE-binding epitope is almost identical with the IgG-binding epitope.     

Further evidence for the alternative pathway of trehalose synthesis linked to maltose utilization in Saccharomyces

Summary Yeast strains bearing a deficiency in trehalose-6-phosphate synthase activity are unable to accumulate trehalose on any carbon source unless they contain one of the MAL genes. If the gene is inducible then synthesis of trehalose occurs specifically during growth on maltose when the MAL gene is constitutive then trehalose accumulation can also be seen when cells are grown on glucose. Different systems for trehalose synthesis were suggested: one of them would require the UDPG-linked trehalose synthase whereas the second would utilize an alternative pathway. We proposed a mechanism by which the gene-product of a MAL gene would serve as a common positive regulator for the expression of the genes coding for maltose permease, -glucosidase and some component of the trehalose accumulation system. In order to elucidate this novel pathway a strain lacking UDPG-linked trehalose synthase activity and harboring a defect in maltose uptake was constructed. Excessive maltose uptake resulted in accumulation of intracellular maltose, and twice as much trehalose as in a control strain. Partial inhibition of hexokinase by xylose affected the ratio between internal maltose and trehalose and significantly reduced glycogen synthesis. Sodium fluoride also blocked glycogen synthesis but allowed for trehalose accumulation. Moreover, a mutant which lacks hexokinase I and II was unable to accumulate trehalose when grown on glucose in spite of the presence of a constitutive MAL2 gene. These results suggest that trehalose synthesis would require G-6-P formation derived from maltose. Such a deviation would allow for slowing down the glycolytic flux which, in turn, would favour efficient maltose utilization. Therefore, trehalose synthesis during growth in media containing glucose serves as an additional parameter for assessing constitutivity of MAL genes.     

Fine Molecular Specificity of Linear and Assembled Antibody Binding Sites in HIV-1 p24

A set of seven murine monoclonal antibodies were generated against a chemically synthesized 11-kDa 104-mer peptide covering the C-terminal residues 270-373 of the p24 gag protein (HIV-1BRU strain). All monoclonal antibodies recognized HIV-1IIIB infected MOLT3 cells by fluorescence and gave positive Western blot signals with viral gag peptides (p55 and/or p24). Oligopeptide binding regions were located with competitive enzyme-linked immunosorbent assays. Detailed epitope scanning analyses (the Geysen technique) were performed by serological testing of the monoclonal antibodies against 99 overlapping hexapeptides which corresponded to the entire 104-mer region. The antibodies bound to p24 peptide sequences located within the 275-293 and 351-368 regions. One antibody (LH104-B) which reacted with residues 357-362 bound to p55 alone. In contrast, another antibody (LH104-I), which recognized the residues 358-363, i.e. with five out of six residues in common with antibody LH104-B for its epitope region, reacted exclusively with p24. At least two of the antibodies (LH104-C and -A) which bound to p24 alone, apparently recognized conformational epitopes. They gave positive reactions with the regions 288-293/351-356 and 284-289/351-356, respectively. This work shows that chemical synthesis of large peptides is a viable alternative approach to immunochemical studies of viral proteins.     

Separate domains of Sendai virus P protein are required for binding to viral nucleocapsids

The role of Sendai virus P protein in viral RNA synthesis involves association with the nucleocapsid template. There is evidence that the carboxyl-terminal region of P protein is responsible for this association (K. W. Ryan and D. W. Kingsbury, 1988, Virology 167, 106-112). To define the P protein sequences involved more precisely, deletions were generated in a cDNA clone of the P gene. Proteins synthesized in vitro from these altered P genes were mixed with extracts from infected cells to determine if they could attach to nucleocapsids. Under conditions where full-size P protein was able to bind, a protein comprising the 95 carboxyl-terminal residues of P protein (Sendai virus X protein) did not bind. This indicated that other P protein residues were required, in addition to the 95 residues at the carboxyl-terminal end. To locate these other residues, P genes were constructed with overlapping deletions of sequences encoding the carboxyl-terminal 40% of the protein. Analysis of these deleted proteins revealed that the necessary residues were in two separate binding domains, amino acids 345 to 412 and 479 to 568 (the carboxyl-terminus). Deletion of the 66 residues between these regions did not affect attachment. Therefore, the formation of a functional binding site requires residues within two separate regions of P protein.     

Immunological characterization of Echinococcus granulosus cyclophilin, an allergen reactive with IgE and IgG4 from patients with cystic echinococcosis

By immunological screening of a cDNA library derived from protoscoleces of Echinococcus granulosus with IgE from patients with cystic echinococcosis (CE) and allergic manifestations, we isolated a protein identical to E. granulosus cyclophilin. The protein, named EA21, has close homology with Malassezia furfur cyclophilin allergen (Mal f 6) and with human cyclophilin. Using immunoblotting (IB) with a polyclonal antibody specific to EA21, we identified E. granulosus cyclophilin both in protoscoleces and in sheep hydatid fluid. Of the 58 sera from patients with CE, 29 (50%) were IgE positive to EA21, whereas, despite the high sequence homology, none were IgE positive to Mal f 6 or human cyclophilin. Only 26 of the 58 patients (45%) had IgG specific to EA21, whereas all patients (100%) had IgG specific to Mal f 6 and human cyclophilin. IB analysis showed that serum IgE-binding reactivity to EA21 differed significantly in patients with and without allergic reactions (20 of 25, 80% versus nine of 33, 27%; P < 10(-4)). Conversely, five of the 25 patients who had CE-related allergic manifestations (20%) and 21 of the 33 who did not (63%) had specific IgG4 (P = 10(-3)) and total IgG to EA21. EA21 induced a proliferative response in 15 of 19 (79%) patients' PBMC regardless of the allergic manifestations, but it induced no IL-4 production. Overall, these findings suggest that E. granulosus cyclophilin is a conserved, constitutive, parasite protein that does not cross-react with cyclophilins from other organisms and is involved in the allergic symptoms related to CE.     

Mutagenesis identifies the critical regions and amino acid residues of suid herpesvirus 1 DNA-binding protein required for DNA binding and strand invasion

Herpesviral DNA-binding protein (DBP) is a unique protein involved in viral DNA replication and genomic recombination. It binds and stabilizes the single-stranded DNA. It also forms the D-loops and promotes the strand invasion. To identify the functional regions and amino acid residues required for DNA binding and D-loop formation, we characterized several DBP mutants of suid herpesvirus 1 (SuHV-1). Acetic anhydride modification assay showed that lysine residues were critical for DNA binding and D-loop formation. Replacement of highly conserved lysine residues with alanine revealed that Lys-756 and Lys-970 were critical for DNA binding, while Lys-161 participated in DNA binding and D-loop formation. Analysis of nested deleted mutants showed that N-terminal 201 amino acid residues and C-terminal 305 amino acid residues were required for D-loop formation and DNA binding, respectively. In conclusion, these findings suggested that SuHV-1 DBP contained critical regions for DNA binding and D-loop formation, and Lys-161, Lys-756, and Lys-970 were required for DNA binding or D-loop formation.