The gene YALI0E20207g from Yarrowia lipolytica encodes an N-acetylglucosamine kinase implicated in the regulated expression of the genes from the N-acetylglucosamine assimilatory pathway

This is an open access article distributed under the terms of the Creative Commons Attribution License.The non-conventional yeast Yarrowia lipolyticapossesses an ORF, YALI0E20207g, which encodes a protein with an amino acid sequence similar to hexokinases fromdifferent organisms. We have cloned that gene and determined several enzymatic properties of its encoded protein showing that it is an N-acetylglucosamine (NAGA) kinase. This conclusion was supported by the lack of growth in NAGA of a strain carrying a YALI0E20207g deletion.We named this gene YlNAG5. Expression of YlNAG5 as well as that of the genes encoding the enzymes of the NAGA catabolic pathway - identified by a BLAST search - was induced by this sugar. Deletion of YlNAG5 rendered that expression independent of the presence of NAGA in the medium and reintroduction of the gene restored the inducibility, indicating that YlNag5 participates in the transcriptional regulation of the NAGA assimilatory pathway genes. Expression of YlNAG5 was increased during sporulation and homozygous Ylnag5/Ylnag5 diploid strains sporulated very poorly as compared with a wild type isogenic control strain pointing to a participation of the protein in the process. Overexpression of YlNAG5 allowed growth in glucose of an Ylhxk1 glk1 double mutant and produced, in a wild type background, aberrant morphologies in different media. Expression of the gene in a Saccharomyces cerevisiae hxk1 hxk2 glk1 triple mutant restored ability to grow in glucose.This work was supported by grant BFU2010-19628-C02-02 from the Spanish Ministry for Science and Innovation (MICINN, now MINECO).Peer Reviewe

The N-acetylglucosamine kinase from Yarrowia lipolytica is a moonlighting protein

Altres ajuts: grant CIVP18A3896 from the Fundación Ramón ArecesIn Yarrowia lipolytica, expression of the genes encoding the enzymes of the N-acetylglucosamine (NAGA) utilization pathway (NAG genes) becomes independent of the presence of NAGA in a Ylnag5 mutant lacking NAGA kinase. We addressed the question of whether the altered transcription was due to a lack of kinase activity or to a moonlighting role of this protein. Glucosamine-6-phosphate deaminase (Nag1) activity was measured as a reporter of NAG genes expression. The NGT1 gene encoding the NAGA transporter was deleted, creating a Ylnag5 ngt1 strain. In glucose cultures of this strain, Nag1 activity was similar to that of the Ylnag5 strain, ruling out the possibility that NAGA derived from cell wall turnover could trigger the derepression. Heterologous NAGA kinases were expressed in a Ylnag5 strain. Among them, the protein from Arabidopsis thaliana did not restore kinase activity but lowered Nag1 activity 4-fold with respect to a control. Expression in the Ylnag5 strain of YlNag5 variants F320S or D214V with low kinase activity caused a repression similar to that of the wild-type protein. Together, these results indicate that YlNag5 behaves as a moonlighting protein. An RNA-seq analysis revealed that the Ylnag5 mutation had a limited transcriptomic effect besides derepression of the NAG genes

Mutations in SCNM1 cause orofaciodigital syndrome due to minor intron splicing defects affecting primary cilia

Orofaciodigital syndrome (OFD) is a genetically heterogeneous ciliopathy characterized by anomalies of the oral cavity, face, and digits. We describe individuals with OFD from three unrelated families having bi-allelic loss-of-function variants in SCNM1 as the cause of their condition. SCNM1 encodes a protein recently shown to be a component of the human minor spliceosome. However, so far the effect of loss of SCNM1 function on human cells had not been assessed. Using a comparative transcriptome analysis between fibroblasts derived from an OFD-affected individual harboring SCNM1 mutations and control fibroblasts, we identified a set of genes with defective minor intron (U12) processing in the fibroblasts of the affected subject. These results were reproduced in SCNM1 knockout hTERT RPE-1 (RPE-1) cells engineered by CRISPR-Cas9-mediated editing and in SCNM1 siRNA-treated RPE-1 cultures. Notably, expression of TMEM107 and FAM92A encoding primary cilia and basal body proteins, respectively, and that of DERL2, ZC3H8, and C17orf75, were severely reduced in SCNM1-deficient cells. Primary fibroblasts containing SCNM1 mutations, as well as SCNM1 knockout and SCNM1 knockdown RPE-1 cells, were also found with abnormally elongated cilia. Conversely, cilia length and expression of SCNM1-regulated genes were restored in SCNM1-deficient fibroblasts following reintroduction of SCNM1 via retroviral delivery. Additionally, functional analysis in SCNM1-retrotransduced fibroblasts showed that SCNM1 is a positive mediator of Hedgehog (Hh) signaling. Our findings demonstrate that defective U12 intron splicing can lead to a typical ciliopathy such as OFD and reveal that primary cilia length and Hh signaling are regulated by the minor spliceosome through SCNM1 activity.This work was supported by a grant from the Spanish Ministry of Science and Innovation (PID2019-105620RB-I00/AEI/10.13039/501100011033)

Disruption of Yarrowia lipolytica TPS1 Gene Encoding Trehalose-6-P Synthase Does Not Affect Growth in Glucose but Impairs Growth at High Temperature

We have cloned the Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase by complementation of the lack of growth in glucose of a Saccharomyces cerevisiae tps1 mutant. Disruption of YlTPS1 could only be achieved with a cassette placed in the 3′half of its coding region due to the overlap of its sequence with the promoter of the essential gene YlTFC1. The Yltps1 mutant grew in glucose although the Y. lipolytica hexokinase is extremely sensitive to inhibition by trehalose-6-P. The presence of a glucokinase, insensitive to trehalose-6-P, that constitutes about 80% of the glucose phosphorylating capacity during growth in glucose may account for the growth phenotype. Trehalose content was below 1 nmol/mg dry weight in Y. lipolytica, but it increased in strains expressing YlTPS1 under the control of the YlTEF1promoter or with a disruption of YALI0D15598 encoding a putative trehalase. mRNA levels of YlTPS1 were low and did not respond to thermal stresses, but that of YlTPS2 (YALI0D14476) and YlTPS3 (YALI0E31086) increased 4 and 6 times, repectively, by heat treatment. Disruption of YlTPS1 drastically slowed growth at 35°C. Homozygous Yltps1 diploids showed a decreased sporulation frequency that was ascribed to the low level of YALI0D20966 mRNA an homolog of the S. cerevisiae MCK1 which encodes a protein kinase that activates early meiotic gene expression

Aislamiento y caracterización de mutantes que suprimen los efectos tóxicos de azúcares sobre mutantes fosfoglicerato mutasa negativos de Saccharomyces cerevisiae

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 10-12-1999La glucosa, al igual que otros azúcares, inhibe el crecimiento de mutantes glícolíticos de levadura en fuentes de carbono alternativas, probablemente por la represión de las enzimas necesarias para la utilización de dichas fuentes. En un intento de identificar genes implicados en la(s) mta(s) de señalización de la presencia de azúcares en Saccharomyces cerevisiae, se han buscado mutaciones supresoras que permitieran crecer a mutantes glicolíticos en presencia de azúcares. Utilizando como material de partida un mutante afectado en el gen GPMI, que codifica la fosfoglicerato mutasa, hemos aislado una serie de mutantes portadores de mutaciones supresoras de los efectos tóxicos de varios azúcares para esta cepa. Una de las mutaciones caracterizadas, que suprimía específicamente la toxicidad de galactosa, resultó afectar al gen GAU, un regulador de la síntesis de los genes GAL. En un mutante ga14 con baja expresión de los genes GAL, el consumo de galactosa es bajo y se libera la represión catabólica de una serie de genes que permiten al mutante original crecer en presencia de este azúcar. Se ha aislado un mutante con una mutación dominante, DDR2-I, que suprime los efectos de glucosa, fnictosa y galactosa. El flujo glicolítico no está afectado por esa mutación. Durante el crecimiento en glicerina+etanol+glucosa, los niveles de metabolitos ensayados en el mutante DDR2-1 fueron similares a los de la cepa parental, excepto en el caso de las triosa fosfatos que eran ligeramente mayores en el mutante. La caracterización del gen responsable de la mutación está en curso. Otros mutantes aislados estaban afectada en la hexokinasa 2 o en la fosfofructokinasa. Estos mutantes resistían el efecto de los tres azúcares antes mencionados

Expression of PEP carboxylase from Escherichia coli complements the phenotypic effects of pyruvate carboxylase mutations in Saccharomyces cerevisiae

We investigated the effects of the expression of the Escherichia coli ppc gene encoding PEP carboxylase in Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. Functional expression of the ppc gene restored the ability of the yeast mutants to grow in glucose-ammonium medium. Growth yield in this medium was the same in the transformed yeast than in the wild type although the growth rate of the transformed yeast was slower. Growth in pyruvate was slowed down in the transformed strain, likely due to a futile cycle produced by the simultaneous action of PEP carboxykinase and PEP carboxylase.This work is part of the project 'From gene to products in yeast: a quantitative approach' supported by the European Community (DGXII Framework IV Program Cell factories). It has benefltted from grant PB94-0091-CO2-01 of the Spanish DGICYT. C.-L. Flores was supported in the initial stages of the work by a Fellowship of the Spanish Instituto de Cooperation IberoamericanaPeer Reviewe

Una proteína moonlighting: la N-acetilglucosamina kinasa de Yarrowia lipolytica controla la expresión de los genes de la vía de utilización de N-acetil-glucosamina

Resumen del póster presentado al XXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Valencia del 7 al 10 de septiembre de 2015.La levadura Y. lipolytica utiliza N-acetil-glucosamina (NAGA) como fuente de carbono. El metabolismo de NAGA se inicia por una fosforilación a NAGA-6P catalizada por la NAGA kinasa. Esta enzima está codifi cada por un gen que hemos clonado y denominado YlNAG5, cuya deleción impide el crecimiento en NAGA. Usando una búsqueda BLAST en la base de datos Génolevures, hemos identifi cado los genes que codifican las enzimas de la vía de utilización de NAGA, que conduce a fructosa-6-P, y hemos determinado que su expresión es inducida por NAGA. Aunque NAGA-6P es también un intermedio en la vía de síntesis de quitina hemos establecido que la expresión de los genes correspondientes a esa vía es independiente de la presencia de NAGA en el medio. La deleción de YlNAG5 hace que los genes de la vía asimilatoria de NAGA se expresen en ausencia de NAGA pero no afecta a la expresión de los genes de la víade síntesis de quitina. La reintroducción de YlNAG5 devuelve la dependencia de NAGA a la expresión de los genes de la vía asimilatoria. Este resultado sugiere que la proteína YlNag5 es una proteína pluriempleada (moonlighting) que además de su función metabólica desempeña otra como reguladora de la transcripción. Se están caracterizando posibles zonas del promotor de YlNAG5 implicadas en la regulación por NAGA kinasa. La proteína YlNag5 está implicada en esporulación. La expresión de YlNAG5 aumenta durante este proceso y un diploide nag5/nag5 esporula pobremente. La secuencia de aminoácidos de la proteína YlNag5 es marcadamente diferente de otras NAGA kinasas. En un árbol filogenético la NAGA kinasa de Y. lipolytica aparece cerca, pero separada, de las escasas NAGA kinasas caracterizadas funcionalmente. Otras proteínas cuya secuencia es relativamente similar a la de la NAGA kinasa de Y. lipolytica anotadas como hexokinasas o sin anotar, podrían ser NAGA kinasas.Trabajo realizado con la ayuda del MICINN (MINECO) BFU2010-19628-C02-02.Peer reviewe

Construction and characterization of a Saccharomyces cerevisiae strain able to grow on glucosamine as sole carbon and nitrogen source

Saccharomyces cerevisiae can transport and phosphorylate glucosamine, but cannot grow on this amino sugar. While an enzyme catalyzing the reaction from glucosamine-6-phosphate to fructose-6-phosphate, necessary for glucosamine catabolism, is present in yeasts using N-acetylglucosamine as carbon source, a sequence homology search suggested that such an enzyme is absent from Saccharomyces cerevisiae. The gene YlNAG1 encoding glucosamine-6-phosphate deaminase from Yarrowia lipolytica was introduced into S. cerevisiae and growth in glucosamine tested. The constructed strain grew in glucosamine as only carbon and nitrogen source. Growth on the amino sugar required respiration and caused an important ammonium excretion. Strains overexpressing YlNAG1 and one of the S. cerevisiae glucose transporters HXT1, 2, 3, 4, 6 or 7 grew in glucosamine. The amino sugar caused catabolite repression of different enzymes to a lower extent than that produced by glucose. The availability of a strain of S. cerevisiae able to grow on glucosamine opens new possibilities to investigate or manipulate pathways related with glucosamine metabolism in a well-studied organism.Te fnancial support by grant CIVP18A3896 of the Fundación Ramón Areces (Madrid, Spain) is gratefully acknowledged.Peer reviewe

Moonlighting Proteins in Yeasts

Proteins able to participate in unrelated biological processes have been grouped under the generic name of moonlighting proteins. Work with different yeast species has uncovered a great number of moonlighting proteins and shown their importance for adequate functioning of the yeast cell. Moonlighting activities in yeasts include such diverse functions as control of gene expression, organelle assembly, and modification of the activity of metabolic pathways. In this review, we consider several well-studied moonlighting proteins in different yeast species, paying attention to the experimental approaches used to identify them and the evidence that supports their participation in the unexpected function. Usually, moonlighting activities have been uncovered unexpectedly, and up to now, no satisfactory way to predict moonlighting activities has been found. Among the well-characterized moonlighting proteins in yeasts, enzymes from the glycolytic pathway appear to be prominent. For some cases, it is shown that despite close phylogenetic relationships, moonlighting activities are not necessarily conserved among yeast species. Organisms may utilize moonlighting to add a new layer of regulation to conventional regulatory networks. The existence of this type of proteins in yeasts should be taken into account when designing mutant screens or in attempts to model or modify yeast metabolism