Triple Mutant Yeast Research Articles

Identification, Expression, and Functional Analysis of the Fructokinase Gene Family in Cassava.

Fructokinase (FRK) proteins play important roles in catalyzing fructose phosphorylation and participate in the carbohydrate metabolism of storage organs in plants. To investigate the roles of FRKs in cassava tuber root development, seven FRK genes (MeFRK1–7) were identified, and MeFRK1–6 were isolated. Phylogenetic analysis revealed that the MeFRK family genes can be divided into α (MeFRK 1, 2, 6, 7) and β (MeFRK 3, 4, 5) groups. All the MeFRK proteins have typical conserved regions and substrate binding residues similar to those of the FRKs. The overall predicted three-dimensional structures of MeFRK1–6 were similar, folding into a catalytic domain and a β-sheet ‘‘lid” region, forming a substrate binding cleft, which contains many residues involved in the binding to fructose. The gene and the predicted three-dimensional structures of MeFRK3 and MeFRK4 were the most similar. MeFRK1–6 displayed different expression patterns across different tissues, including leaves, stems, tuber roots, flowers, and fruits. In tuber roots, the expressions of MeFRK3 and MeFRK4 were much higher compared to those of the other genes. Notably, the expression of MeFRK3 and MeFRK4 as well as the enzymatic activity of FRK were higher at the initial and early expanding tuber stages and were lower at the later expanding and mature tuber stages. The FRK activity of MeFRK3 and MeFRK4 was identified by the functional complementation of triple mutant yeast cells that were unable to phosphorylate either glucose or fructose. The gene expression and enzymatic activity of MeFRK3 and MeFRK4 suggest that they might be the main enzymes in fructose phosphorylation for regulating the formation of tuber roots and starch accumulation at the tuber root initial and expanding stages.

Genome-Wide Quantitative Fitness Analysis (QFA) of Yeast Cultures.

We provide a detailed protocol for robot-assisted, genome-wide measurement of fitness in the model yeast Saccharomyces cerevisiae using Quantitative Fitness Analysis (QFA). We first describe how we construct thousands of double or triple mutant yeast strains in parallel using Synthetic Genetic Array (SGA) procedures. Strains are inoculated onto solid agar surfaces by liquid spotting followed by repeated photography of agar plates. Growth curves are constructed and the fitness of each strain is estimated. Robot-assisted QFA, can be used to identify genetic interactions and chemical sensitivity/resistance in genome-wide experiments, but QFA can also be used in smaller scale, manual workflows.

Antibodies elicited by yeast glycoproteins recognize HIV-1 virions and potently neutralize virions with high mannose N-glycans

The glycan shield on the human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein has drawn attention as a target for HIV-1 vaccine design given that an increasing number of potent and broadly neutralizing antibodies (bNAbs) recognize epitopes entirely or partially comprised of high mannose type N-linked glycans. In an attempt to generate immunogens that target the glycan shield of HIV-1, we previously engineered a triple mutant (TM) strain of Saccharomyces cerevisiae that results in exclusive presentation of high mannose type N-glycans, and identified five TM yeast glycoproteins that support strong binding of 2G12, a bNAb that targets a cluster of high mannose glycans on the gp120 subunit of Env. Here, we further analyzed the antigenicity and immunogenicity of these proteins in inducing anti-HIV responses. Our study demonstrated that the 2G12-reactive TM yeast glycoproteins efficiently bound to recently identified bNAbs including PGT125-130 and PGT135 that recognize high mannose glycan-dependent epitopes. Immunization of rabbits with a single TM yeast glycoprotein (Gp38 or Pst1), when conjugated to a promiscuous T-cell epitope peptide and coadministered with a Toll-like receptor 2 agonist, induced glycan-specific HIV-1 Env cross-reactive antibodies. The immune sera bound to both synthetic mannose oligosaccharides and gp120 proteins from a broad range of HIV-1 strains. The purified antibodies recognized and captured virions that contain both complex- and high mannose-type of N-glycans, and potently neutralized virions from different HIV-1 clades but only when the virions were enforced to retain high mannose N-glycans. This study provides insights into the elicitation of anti-carbohydrate, HIV-1 Env-cross reactive antibodies with a heterologous glycoprotein and may have applications in the design and administration of immunogens that target the viral glycan shield for development of an effective HIV-1 vaccine.

Identification and structure of six members of the hexokinase gene family in Vitis vinifera: Cloning, expression, and functional analysis of a putative chloroplast stromal-type hexokinase

SummaryHexokinase catalyses the first step in the metabolism of glucose, but has also been proposed to be involved in sugar sensing and signalling in both yeasts and plants. The substrates of hexokinase such as glucose (Glc) and fructose (Fruc) are also the most important sugars during grape berry ripening. The grapevine proteome database was analysed to investigate the roles of hexokinases in grape berry growth and development. Six hexokinase genes displaying high nucleotide sequence identity (72 – 87%) with hexokinase genes in other species were identified. Most of the Vitis vinifera hexokinase (VvHXK) genes had a highly conserved genomic structure consisting of nine exons and eight introns. A search for cis–regulatory elements in the promoter regions of all six hexokinase genes revealed that most were probably regulated by light, sugar, phytohormones, or abiotic stress. The isolation and expression of a hexokinase cDNA from V. vinifera ‘Sultanine’, named VvHXK3, was also reported. Analysis of the predicted amino acid sequence of VvHXK3 suggested that the protein could be a chloroplast-stromal hexokinase with a possible transit peptide cleavage site after amino acid residue 26. The VvHXK3 gene was differentially expressed in a variety of organs including berries, leaves, roots, and pollen, but its expression was highest in berries during their early development and at the start of ripening. To determine its function, VvHXK3 cDNA was expressed in a triple mutant yeast strain that lacked the ability to phosphorylate Glc and Fru and, therefore, was unable to grow on these sugars as sole carbon source. Mutant yeast cells that expressed VvHXK3 grew on both Glc and Fru, indicating that VvHXK3 could complement this mutant and had hexokinase activity.

2G12/PGT-binding yeast glycoprotein Gp38 elicits mannose-specific HIV-1 Env cross-reactive antibodies

Results Using the newly identified, glycan-specific PGT bNAbs to search for their binders from a triple mutant(TM) strain of Saccharomyces cerevisiae, we found that the PGT bNAbs not only bind to the previously identified 2G12reactive glycoproteins but also recognize several unknown proteins in TM yeast. One of them was identified as a short version of Gp38 with N-terminus truncation. Based on immunization of rabbits with various formulations and strategies, we found that a high titer of HIV-1 Env cross-reactive antibodies was induced when using a promiscuous T-cell epitope peptide conjugated Gp38 in a formulation with a Toll-like receptor 2 agonist and aluminum salts. The Gp38-elicited antibodies could bind to a broad range of monomeric gp120s from HIV and SIV. Moreover, the antibodies could also efficiently neutralize HIV-1 pseudoviruses when the viruses were produced in the presence of a mannosidase inhibitor kifunensine, which enriches high-mannose Man9GlcNAc2 N-linked glycans. Glycan microarray analysis showed that these antibodies bind to the synthetic Mana1,2-Mana1,2-Man containing oligosaccharides.

Yeast-Elicited Cross-Reactive Antibodies to HIV Env Glycans Efficiently Neutralize Virions Expressing Exclusively High-Mannose N-Linked Glycans

The HIV envelope (Env) protein uses a dense coat of glycans to mask conserved domains and evade host humoral immune responses. The broadly neutralizing antibody 2G12, which binds a specific cluster of high-mannose glycans on HIV Env, shows that the glycan shield can also serve as a target for neutralizing antibodies. We have described a triple mutant Saccharomyces cerevisiae strain that expresses high-mannose glycoproteins that bind to 2G12. When used to immunize rabbits, this yeast elicits antibodies that bind to gp120-associated glycans but fail to neutralize virus. Here we sought to determine the reason for these discordant results. Affinity purification of sera over columns conjugated with three 2G12-reactive yeast glycoproteins showed that these proteins could adsorb 80% of the antibodies that bind to gp120 glycans. Despite binding to monomeric gp120, these mannose-specific antibodies failed to bind cell surface-expressed trimeric Env. However, when Env was expressed in the presence of the mannosidase inhibitor kifunensine to force retention of high-mannose glycans at all sites, the purified antibodies gained the abilities to bind trimeric Env and to strongly and broadly neutralize viruses produced under these conditions. Combined, these data show that the triple mutant yeast strain elicits antibodies that bind to high-mannose glycans presented on the HIV envelope, but only when they are displayed in a manner not found on native Env trimers. This implies that the underlying structure of the protein scaffold used to present the high-mannose glycans may be critical to allow elicitation of antibodies that recognize trimeric Env and neutralize virus.

Cloning, expression and characterization of LeFRK3, the fourth tomato (Lycopersicon esculentum Mill.) gene encoding fructokinase

A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate recognition sites and an ATP-binding domain. Confirmation for its fructokinase activity was obtained by complementation of triple mutant yeast cells that are unable to phosphorylate or grow on either glucose or fructose as LeFRK3 complemented growth on fructose but not on glucose. Moreover, soluble crude protein extracts prepared from the transformed yeast cells revealed fructose but not glucose phosphorylation activity. In contrast to the LeFRK1 gene product which is inhibited neither by fructose nor by Mg, and to LeFRK2 gene product which is inhibited by both fructose and Mg, the LeFRK3 product is inhibited by fructose but not by Mg. Separation by HPLC-ion exchange chromatography pointed to the gene product of LeFRK3 as the protein responsible for the third peak of fructokinase activity (FKIII), sharing the same pattern of fructose inhibition previously identified with FKIII in tomato fruits. Mapping of tomato fructokinases indicated that LeFRK3 is located on chromosome 2, unlike LeFRK1 (chromosome 3), LeFRK2 (chromosome 6), and LeFRK4 (chromosome 10). The relative expression levels of the four known FRK genes in different tomato organs were analyzed by quantitative RT-PCR. LeFRK2 and LeFRK3 are the predominant genes expressed in all organs with LeFRK3 having the highest level of expression in leaves and apices. LeFRK4 is expressed only in stamens. This differential expression patterns combined with the different biochemical characteristics of the four FRK isozymes suggest that each plays a different role in plant development.

Cloning and characterization of a cDNA encoding hexokinase from tomato

Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransferase, EC 2.7.1.1) were isolated from tomato ( Lycopersicon esculentum) by RT-PCR using degenerate primers. Southern blot analysis suggested the existence of two divergent HXK genes. A complete cDNA of one HXK was isolated by screening a cDNA library prepared from young cherry tomato fruit. The 1770 bp cDNA of LeHXK2 contained an open reading frame encoding a 496 amino acid protein that has 69% identity with the two Arabidopsis HXKs, 83 and 85% identity with potato StHXK1 and tobacco NtHXK, respectively. However, this clone had 97% amino acid identity with potato StHXK2 and, therefore, was named LeHXK2. LeHXK2 cDNA was expressed in a triple mutant yeast ( Saccharomyces cerevisiae) strain which lacked the ability to phosphorylate glucose and fructose and, therefore, was unable to grow on these sugars as carbon sources. Mutant cells expressing LeHXK2 grew on both glucose and fructose with shorter doubling time on glucose. The kinetic properties of LeHXK2 expressed in yeast were determined after the purification of LeHXK2 by HPLC-ion exchange chromatography, confirming the identity of LeHXK2 as hexokinase with higher affinity to glucose. LeHXK2 mRNA was detected by RT-PCR expression analysis in all organs and tissues and at all stages of fruit development. However, semi-quantitative RT-PCR analysis showed that LeHXK2 was most highly expressed in flowers.

Production of Human Compatible High Mannose-type (Man5GlcNAc2) Sugar Chains in Saccharomyces cerevisiae

A yeast mutant capable of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. An expression vector for alpha-1,2-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. An in vitro alpha-1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an alpha-1, 2-mannosidase expression vector. The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man5GlcNAc2. This is the first report of a recombinant S. cerevisiae able to produce Man5GlcNAc2-oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.