Yeast Enzyme Research Articles

Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells

Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 μm (yeast PGK1) and 6.86 μm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 μm (yeast PGK1) and 186 μm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 μm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.

The single CCA-adding enzyme of T. brucei has distinct functions in the cytosol and in mitochondria

tRNAs universally carry a CCA nucleotide triplet at their 3'-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.

Structural Insight into the Mechanism of N-Linked Glycosylation by Oligosaccharyltransferase.

Asparagine-linked glycosylation, also known as N-linked glycosylation is an essential and highly conserved post-translational protein modification that occurs in all three domains of life. This modification is essential for specific molecular recognition, protein folding, sorting in the endoplasmic reticulum, cell–cell communication, and stability. Defects in N-linked glycosylation results in a class of inherited diseases known as congenital disorders of glycosylation (CDG). N-linked glycosylation occurs in the endoplasmic reticulum (ER) lumen by a membrane associated enzyme complex called the oligosaccharyltransferase (OST). In the central step of this reaction, an oligosaccharide group is transferred from a lipid-linked dolichol pyrophosphate donor to the acceptor substrate, the side chain of a specific asparagine residue of a newly synthesized protein. The prokaryotic OST enzyme consists of a single polypeptide chain, also known as single subunit OST or ssOST. In contrast, the eukaryotic OST is a complex of multiple non-identical subunits. In this review, we will discuss the biochemical and structural characterization of the prokaryotic, yeast, and mammalian OST enzymes. This review explains the most recent high-resolution structures of OST determined thus far and the mechanistic implication of N-linked glycosylation throughout all domains of life. It has been shown that the ssOST enzyme, AglB protein of the archaeon Archaeoglobus fulgidus, and the PglB protein of the bacterium Campylobactor lari are structurally and functionally similar to the catalytic Stt3 subunit of the eukaryotic OST enzyme complex. Yeast OST enzyme complex contains a single Stt3 subunit, whereas the human OST complex is formed with either STT3A or STT3B, two paralogues of Stt3. Both human OST complexes, OST-A (with STT3A) and OST-B (containing STT3B), are involved in the N-linked glycosylation of proteins in the ER. The cryo-EM structures of both human OST-A and OST-B complexes were reported recently. An acceptor peptide and a donor substrate (dolichylphosphate) were observed to be bound to the OST-B complex whereas only dolichylphosphate was bound to the OST-A complex suggesting disparate affinities of two OST complexes for the acceptor substrates. However, we still lack an understanding of the independent role of each eukaryotic OST subunit in N-linked glycosylation or in the stabilization of the enzyme complex. Discerning the role of each subunit through structure and function studies will potentially reveal the mechanistic details of N-linked glycosylation in higher organisms. Thus, getting an insight into the requirement of multiple non-identical subunits in the N-linked glycosylation process in eukaryotes poses an important future goal.

Production and Thermal Characterization of Ethanol Blends from Black Jaggery

To protect the environment from the global warming dependency on the fossil fuels have to be reduced. Locally available alternate fuels are greatly prominent for the development of industrialization compared to conventional fuels. This paper mainly deals with the production of Ethanol from a source called “Black Jaggery” and an Optimization of the extracted alcohol to attain the characteristics and properties which would be essential to blend the alcohol with an existing fossil fuel. Black Jaggery being a sugar-based product is fermented in the presence of a yeast enzyme for several days and is distilled to extract the bio-fuel (ethanol) from the source. The extracted oil is characterized for the thermal properties by using thermal constant analyzer TPS-500 which will be helpful for the combustion studies. Obtained results shows that compared to E-5, E-10 and E-20, E-15 blend shows better thermal properties increased thermal conductivity, thermal diffusivity with reduced specific heat.

Treatment and optimization of unconventional heating to enhance the printability of Rami fabric by using Brewer’s Yeast enzyme

Treatment and optimization of a non-traditional heating to enhancement the printability of Rami fabric by using Brewer’s Yeast enzyme was studied. The treatment of raw and semi-finished ramie fabrics was submitted to innovative treatment using microwave irradiation and under a variety of conditions. Variables studies including yeast concentration, duration of treatment, and treated temperature to optimize the treatment condition. Conditional changes in the innovatively treated fabric vis-à-vis those of untreated fabric were presented. The obtained results showed that the innovative treatment process using microwave irradiation consumes less time and energy. Besides that, there is an enhancement of physical and chemical properties of fabrics under study, which leads to enhancement of its printability with the reactive dye. The treated, as well as untreated fabrics, were characterized by using scanning electron microscopy (SEM) coupled with Fourier transforms infrared spectroscopy (FTIR). The effect of treatment with Brewer’s Yeast enzyme coupled with microwave irradiation on physical and mechanical properties was investigated by using X-ray analysis. The effects of treatment with yeast enzyme on the multifunctional properties of the fibers including coloration, and antibacterial activity for E. coli as an example for gram-negative and S. aureus as an example of gram-positive bacteria were evaluated. The overall results point out that, the treated fabrics exhibited excellent color fastness as well as good antibacterial if compared to the untreated fabrics, in straightforward, the procedure adopted for fabricating these multifunctional ramie fabrics is environmentally friendly beside time and energy-saving.

In silico and in vitro comparison of nicotinamide adenine dinucleotide phosphate dependent xylose reductase rossmaan fold in Debaryomycetaceae yeast family

abstract Meyerozyma caribbica is prominently used for the production of xylitol from agro waste hydrolysate of the corncob. M. caribbica was isolated from fermented apple juice, the nicotinamide adenine dinucleotide phosphate (NADP) dependent xylose reductase (XR) enzyme act as a catalyst for the production of xylitol from xylose during xylose fermentation by M. caribbica. The enzyme XR was extracted, partially purified, sequenced and molecular weight was determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). This study explores the in silico and in vitro comparative analysis of the cofactor NADP binding mechanism in rossmann fold of isolated M. caribbica and other xylose fermenting Debaryomycetaceae family yeast. The rossmaan fold analysis of M. caribbica and Debaryomycetaceae family of yeast revealed, xylose fermenting organisms such as Scheffersomyces stipitis, Debaryomyces hansenii, Debaryomyces nepalensis, Candida parapsilosis, Candida maltosa have divergence structure of rosmann fold. Whereas M. caribbica and Candida tenuis has unique structure of rossmann fold. Also, the identified rossmann fold of Debaryomycetaceae family yeast and isolated M. caribbica are conserved to interact with cofactor NADP by H2 bonding, which displays the proximity evolutionary relationship of the enzyme in Debaryomycetaceae family yeast. Annotation analysis showed the isolated M. caribbica has the ability of aldopentose fermentation for xylitol production.

Use of Agro-Industrial Wastes for the Production of a Wild Yeast Enzyme with Disintegration Activity on Plant Tissues

The objective of the present study was to develop a cost-effective medium, using agro-industrial wastes for the production of a polygalacturonase by Wickerhanomyces anomalus of interest in cassava starch industries. The effect of several raw agro-industrial wastes and others nutrients on polygalacturonase production by W. anomalus, were evaluated, in a reference fermentation medium, using statistical designs, by batch culture. The ability of the cell-free supernatant to extract cassava starch was evaluated. Lemon peel was the best inducer for the production of PGase. Statistical analysis of the data showed that lemon peel, Mg+2 and PO4HK2 had significant effect on PGase production, and the others variables (yeast extract, Ca+2, Fe+2, amino acid and trace element solution) were no significant. PGase synthesis reached ~31 EUmL-1, in the OFM (glucose, lemon peel, urea, vitamins, KH2PO4 and MgSO4), after 12 h of culture, at a lab scale bioreactor. PGase of W. anomalus, was able to disintegrate cassava tuber tissue, and the starch granules contained within the cells were released into the reaction medium. Lemon peel can be used as inducer for PGase production by W. anomalus, in a low cost culture medium, appropriate for the production of the enzyme at large scale.

Assessment of the suitability of dry yeast for the production of wines and wine beverages from sea buckthorn

Biochemical transformations under the action of the yeast enzyme complex affect not only the utilization of sugar to the final product - ethyl alcohol, but also secondary processes accompanied by the synthesis of minor side components that have a significant impact on the bouquet and taste of finished drinks. Modern dry yeast for winemaking produced industrially is characterized by significant variability in characteristics, which requires testing of their use on various types of raw materials. In the experiment, data were obtained on the use of dry yeast produced by LALLEMAND Inc. company (Canada) for the production of sea buckthorn wines and wine beverages. It is established that the studied yeast samples allow to obtain a finished product with different characteristics, which makes it possible to differentially approach the industrial implementation of the technology of wines and wine drinks from sea buckthorn by varying the yeast strains.

Highly Efficient Synthesis of Glutathione via a Genetic Engineering Enzymatic Method Coupled with Yeast ATP Generation

Glutathione is a tripeptide compound with many important physiological functions. A new, two-step reaction system has been developed to efficiently synthesize glutathione. In the first step, glutamate and cysteine are condensed to glutamyl-cysteine by endogenous yeast enzymes inside the yeast cell, while consuming ATP. In the second step, the yeast cell membrane is lysed by the permeabilizing agent CTAB (cetyltrimethylammonium bromide) to release the glutamyl-cysteine, upon which added glutathione synthetase converts the glutamyl-cysteine and added glycine into glutathione. The ATP needed for this conversion is supplied by the permeabilized yeast cells of glycolytic pathway. This method provided sufficient ATP, and reduced the feedback inhibition of glutathione for the first-step enzymatic reaction, thereby improving the catalytic efficiency of the enzyme reaction. In addition, the formation of suitable oxidative stress environment in the reaction system can further promote glutathione synthesis. By HPLC analysis of the glutathione, it was found that 2.1 g/L reduced glutathione is produced and 17.5 g/L oxidized glutathione. Therefore, the new reaction system not only increases the total glutathione, but also facilitates the subsequent separation and purification due to the larger proportion of oxidized glutathione in the reaction system.

Citrus limetta peels: a promising substrate for the production of multienzyme preparation from a yeast consortium

Citrus limetta peels (CLP), a waste material generated by juice industries, has scarcely been reported for the production of yeast enzymes. The study was conducted to obtain a multienzyme preparation from a yeast consortium under solid-state fermentation of CLP. The substrate, CLP, was pretreated using either acid or alkali, and factors affecting production of multienzyme were studied by generating two separate Plackett–Burman designs. Since, alkali-pretreated CLP yielded higher titers; therefore, significant factors affecting multienzyme preparation using this substrate were optimized by employing Box–Behnken design. The analysis revealed that under optimized conditions, i.e., cultivation of yeast strains for 72 h to alkali-pretreated CLP moistened with mineral salt medium having pH 5 yielded more than 10 IU mL−1 of cellulase, xylanase, and amylase. The multienzyme was studied for its application to saccharify fruit and non-fruit wastes and for orange juice clarification. The data showed that the enzyme preparation could release 3.03 mg L−1 h−1 of reducing sugars from various crude substrates and was able to reduce turbidity of orange juice by 11% with substantial decrease in viscosity and acidity. Hence, CLP appeared as a promising substrate to produce multienzyme preparation from yeast consortium.

Исследование пектолитической способности дрожжей Saccharomyces Cerevisiae

Научный и практический интерес представляет поиск современных биотехнологических приемов получения новых пребиотических веществ, в частности пектиновых олигосахаридов (ПОС). ПОС получают расщеплением макромолекулы пектина химическими или биохимическими способами. ПОС имеют большой потенциал использования в пищевых и кормовых целях. В работе экспериментально доказана пектолитическая способность дрожжей Saccharomyces Cerevisiae, обеспечивающая возможность получения жизнеобеспечивающего источника энергии для их роста из раствора яблочного пектина. Питательной средой и растворителем пектина служила натуральная творожная пастеризованная сыворотка. В рабочей гипотезе энергетического использования пектина ключевое место отводится ферментам дрожжей, расщепляющим полигалактуроновую цепь пектина эндополигалактуроназе и пектин-лиазе, при одновременно протекающем процессе деметилирования пектина пектинэстераразой. Приведены экспериментальные данные в подтверждение указанной гипотезы. Полученные результаты свидетельствуют об использовании продуктов расщепления пектина дрожжами по типу дыхания. Результаты исследования подтверждают наличие пектинолитической активности у дрожжей Saccharomyces Cerevisiae, что обеспечивает расщепление пектина до уровня пектиновых олигосахаридов (ПОС) и использование отдельных мономеров полигалактуроновой цепи в энергетическом обмене дрожжей.

Biochemistry in the context of Beer Science

Biochemistry contents are essentials to understanding brewing and students often fail to connect theory with practical applications, in this context, the discipline Beer Science was created, complimenting students` formation. The objective was to integrate the knowledge of biochemistry in beer production and evaluate the students' perception of importance of these. The discipline was given in 2017 and 2018, 24 students per class, with the prerequisite being Biochemistry approval. A didactic sequence was created, starting from the historical aspects and ending with a real beer production. Mains concepts: ph and buffer system; carbohydrates, proteins and lipids in the malt; hops oils/composition; malt and yeast enzymes; fermentative metabolism etc. A questionnaire was applied at the end of the course, evaluating students' perceptions about it, in addition, they delivered a group work simulating a production. Besides the lectures and exercises, 7 practical classes were created, involving the supplies characteristics, mashing buffer effect, temperature and pH effect on the mashing enzymes. There were 31 responses and the students felt very motivated and all considered the biochemical knowledges vital for brewing understanding, attributing greater importance to enzymes and fermentations knowledges, sequentially, to pH, buffer and carbohydrates structure and metabolism. The students emphasized the role of practical classes in order to establish the connections between biochemistry theory and beer production, highlighting the mashing comparison from a matrix of two pH and three temperatures, evaluating the starch hydrolysis with lugol, the amount of fermentable substrate by benedict reaction, and the density by light refractometer. The simulations evaluation indicated that practically all groups were able to apply theoretical knowledge. It is noteworthy that most students felt capable of brewing beer. Disciplines like this are fundamental for the connection between biochemical knowledge and practical applications, such as brewing, especially when developing practices that make this integration possible.

A switch element in the autophagy E2 Atg3 mediates allosteric regulation across the lipidation cascade

Autophagy depends on the E2 enzyme, Atg3, functioning in a conserved E1-E2-E3 trienzyme cascade that catalyzes lipidation of Atg8-family ubiquitin-like proteins (UBLs). Molecular mechanisms underlying Atg8 lipidation remain poorly understood despite association of Atg3, the E1 Atg7, and the composite E3 Atg12–Atg5-Atg16 with pathologies including cancers, infections and neurodegeneration. Here, studying yeast enzymes, we report that an Atg3 element we term E123IR (E1, E2, and E3-interacting region) is an allosteric switch. NMR, biochemical, crystallographic and genetic data collectively indicate that in the absence of the enzymatic cascade, the Atg3E123IR makes intramolecular interactions restraining Atg3′s catalytic loop, while E1 and E3 enzymes directly remove this brace to conformationally activate Atg3 and elicit Atg8 lipidation in vitro and in vivo. We propose that Atg3′s E123IR protects the E2~UBL thioester bond from wayward reactivity toward errant nucleophiles, while Atg8 lipidation cascade enzymes induce E2 active site remodeling through an unprecedented mechanism to drive autophagy.

Inhibition of bacterial ubiquitin ligases by SidJ-calmodulin catalysed glutamylation.

The family of bacterial SidE enzymes catalyzes phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophilia, a pathogenic bacterium causing Legionnaires’ disease1,2,3. SidEs share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes their toxicity in yeast and mammalian cells, through an unknown mechanism4–6. Deletion of SidJ leads to a significant defect in the growth of Legionella in both its natural host amoeba and in murine macrophages4,5. Here, we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADPribosyl transferase (mART) domain of SdeA thus blocking its ubiquitin (Ub) ligase activity. SidJ glutamylation activity requires interaction with Calmodulin (CaM), a eukaryotic specific co-factor, and can be regulated by intracellular changes in Ca2+ concentrations. The cryo-EM structure of SidJ/human apo-CaM complex revealed the architecture of this unique heterodimeric glutamylase. In infected cells, we show that SidJ mediates glutamylation of SidEs on the surface of Legionella-containing vacuoles (LCVs). Using quantitative proteomics, we also uncovered multiple host proteins as putative targets of SidJ-mediated glutamylation. Collectively, this study reveals the mechanism of SidE ligases inhibition by a SidJ/CaM glutamylase and opens new avenues for studying protein glutamylation, an understudied protein modification in higher eukaryotes.

Outline of the biosynthesis and regulation of ergosterol in yeast.

Sterols are crucial functional components for eukaryotic cell membrane. Due to versatile activities, sterols show wide applications in food and pharmaceutical industries. Ergosterol not only reflects cell growth but also serves as the precursor for manufacturing steroid drugs. To date, the ergosterol biosynthetic pathway in yeast has been reported, and the industrial production of ergosterol is achieved by yeast fermentation or extraction from fungal mycelia. Here, we summarize its biosynthesis, regulation, transportation, and subcellular location of enzymes in yeast. In particular, we review the regulation of ergosterol biosynthesis at transcriptional, translational and post-translational levels. Furthermore, we advocate metabolic engineering and fermentation strategies for high-level production of ergosterol. This study may provide evaluable insights into metabolic engineering of yeast for scaled-up fermentation production of ergosterol or beyond.

Ppn2 endopolyphosphatase overexpressed in Saccharomyces cerevisiae: Comparison with Ppn1, Ppx1, and Ddp1 polyphosphatases

Saccharomyces cerevisiae has high level of inorganic polyphosphate and a multicomponent system of its metabolism, including polyphosphatases Ppx1, Ppn1, Ddp1, and Ppn2.The aim of the study was to construct the yeast strain overexpressing Ppn2 and to compare the properties of Ppn2, Ppx1, Ppn1, and Ddp1 purified from overexpressing strains of S. cerevisiae. We overexpressed Ppn2 in S. cerevisiae under a strong constitutive promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase-encoding gene and suggested biochemical criteria for distinguishing among yeast polyphosphatases, which is important for their identification and understanding of their functions. Ppn2, Ppn1, and Ddp1 had endopolyphosphatase activities, whereas Ppx1 did not. Ppx1 and Ppn1 exhibited high and Ddp1 and Ppn2 low exopolyphosphatase activity: 240, 500, 0.05 and 0.1 U/mg protein, respectively. The enzymes had distinct patterns of exopolyphosphatase activities stimulation by divalent metal ions. Ppn2, Ppn1 and Ddp1 displayed endopolyphosphatase activity in the presence of 1 mM Mg2+. The endopolyphosphatase activities of Ppn2 and Ppn1 were induced by 0.01 mM of Co2+ or Zn2+, whereas that of Ddp1 required 0.1 mM of these cations. The endopolyphosphatase activity of Ppn1 was inhibited by 0.01 mg mL−1 of heparin, while endopolphosphatase activity of Ppn2 was weakly sensitive to 0.25 mg mL−1 of heparin. The Ppx1 and Ppn1 activity with guanosine tetraphosphate was nearly 80% of activity with long-chain polyphosphates. The Ppn1 hydrolyzed dATP, while Ppx1 did not. The differences in the mode of polyphosphate hydrolysis, substrate specificity, metal ion dependence and cell localization suggest distinct roles of these enzymes in yeast.

Functional expression and activity screening of all human cytochrome P450 enzymes in fission yeast.

Here, a complete set of recombinant fission yeast strains that coexpress each of the 57 human cytochrome P450 (CYP) enzymes together with their natural human electron transfer partner(s) was cloned. This strain collection was tested with two luminogenic probe substrates, and 31 human CYPs (including the orphan enzymes CYP2A7, CYP4A22 and CYP20A1) were found to metabolize at least one of these. Since other substrates are known for the remaining enzymes, all human CYPs are now shown to be active. Interestingly, CYP5A1 was found for the first time to work on a substrate other than prostaglandin H2 , and, moreover, to catalyze an aliphatic hydroxylation reaction that consumes molecular oxygen. Also, the ability of CYP11A1 to catalyze an aryl hydroxylation is another unexpected result.

Limits to Compensatory Mutations: Insights from Temperature-Sensitive Alleles.

Previous experiments with temperature-sensitive mutants of the yeast enzyme orotidine 5'-phosphate decarboxylase (encoded in gene URA3) yielded the unexpected result that reversion occurs only through exact reversal of the original mutation (Jakubowska A, Korona R. 2009. Lack of evolutionary conservation at positions important for thermal stability in the yeast ODCase protein. Mol Biol Evol. 26(7):1431-1434.). We recreated a set of these mutations in which the codon had two nucleotide substitutions, making exact reversion much less likely. We screened these double mutants for reversion and obtained a number of compensatory mutations occurring at alternative sites in the molecule. None of these compensatory mutations fully restored protein performance. The mechanism of partial compensation is consistent with a model in which protein stabilization is additive, as the same secondary mutations can compensate different primary alternations. The distance between primary and compensatory residues precludes direct interaction between the sites. Instead, most of the compensatory mutants were clustered in proximity to the catalytic center. All of the second-site compensatory substitutions occurred at relatively conserved sites, and the amino acid replacements were to residues found at these sites in a multispecies alignment of the protein. Based on the estimated distribution of changes in Gibbs free energy among a large number of amino acid replacements, we estimate that, for most proteins, the probability that a second-site mutation would have a sufficiently large stabilizing effect to offset a temperature-sensitive mutation in the order of 10-4 or less. Hence compensation is likely to take place only for slightly destabilizing mutations because highly stabilizing mutations are exceeding rare.

Allosteric activation of yeast enzyme neutral trehalase by calcium and 14-3-3 protein.

Neutral trehalase 1 (Nth1) from Saccharomyces cerevisiae catalyzes disaccharide trehalose hydrolysis and helps yeast to survive adverse conditions, such as heat shock, starvation or oxidative stress. 14-3-3 proteins, master regulators of hundreds of partner proteins, participate in many key cellular processes. Nth1 is activated by phosphorylation followed by 14-3-3 protein (Bmh) binding. The activation mechanism is also potentiated by Ca(2+) binding within the EF-hand-like motif. This review summarizes the current knowledge about trehalases and the molecular and structural basis of Nth1 activation. The crystal structure of fully active Nth1 bound to 14-3-3 protein provided the first high-resolution view of a trehalase from a eukaryotic organism and showed 14-3-3 proteins as structural modulators and allosteric effectors of multi-domain binding partners.

Engineered SUMO/protease system identifies Pdr6 as a bidirectional nuclear transport receptor.

Cleavage of affinity tags by specific proteases can be exploited for highly selective affinity chromatography. The SUMO/SENP1 system is the most efficient for such application but fails in eukaryotic expression because it cross-reacts with endogenous proteases. Using a novel selection system, we have evolved the SUMOEu/SENP1Eu pair to orthogonality with the yeast and animal enzymes. SUMOEu fusions therefore remain stable in eukaryotic cells. Likewise, overexpressing a SENP1Eu protease is nontoxic in yeast. We have used the SUMOEu system in an affinity-capture-proteolytic-release approach to identify interactors of the yeast importin Pdr6/Kap122. This revealed not only further nuclear import substrates such as Ubc9, but also Pil1, Lsp1, eIF5A, and eEF2 as RanGTP-dependent binders and thus as export cargoes. We confirmed that Pdr6 functions as an exportin in vivo and depletes eIF5A and eEF2 from cell nuclei. Thus, Pdr6 is a bidirectional nuclear transport receptor (i.e., a biportin) that shuttles distinct sets of cargoes in opposite directions.