Laboratory of Carlos Gancedo Rodríguez

Research topics

N-acetylglucosamine kinase from Yarrowia lipolytica as a moonlighting protein


N-acetylglucosamine kinase from Yarrowia lipolytica as a moonlighting protein


N-acetylglucosamine kinase from Yarrowia lipolytica as a moonlighting protein


 

Moonlighting/N-acetylglucosamine/Yarrowia/Yeast/Interaction/ transcription/ Glucosamine

 

 

Moonlighting proteins are a subset of the multifunctional proteins group. Curently there are about 400 proteins identifies in that subset. Results from our group have identified the N-acetylglucosamine (NAGA) kinase from Yarrowia lipolytica as a potential moonlighting protein. We are working to verify this idea and, if positive, identify interactions of the protein with other components of the cell.

 - A first objective is to establish if the catalytic activity of the NAGA kinase is necessary for the regulatory function of this enzyme. To approach this we cloned genes encoding NAGA kinases from Homo sapiens, Candida albicans, Magnaporthe grisea and Arabidopsis thaliana and inserted them in adequate plasmids. Mutants of Y. lipolytica lacking NAGA kinase (nag5) were transformed with these plasmids and both the complementation of the growth phenotype and the transcription of the genes of the NAGA catabolic pathway were examined in the transformants.

 - We are trying to localize sites that may affect the putative non-canonical activity of the protein. This is not straightforward the amino acid sequence of YlNag5 has no strong similarity with that of other NAGA kinases. However, we have identified a conserved site in a region of conserved structure whose mutation in the mammalian protein abolishes its kinase activity but does not affect its regulatory role in dendritic arborization. We are performing the mutation of this site to study its effect on the yeast protein.

 - We have generated an antibody against the NAGA kinase from Y. lipolytica to study its intracellular localization and its possible interactions with other proteins.

 - In the catabolism of NAGA glucosamine-6-phosphate in Y. lipolytica appears as an intermediary metabolite. In the model yeast Saccharomyces cerevisiae this metabolite is generated in the presence of glucosamine, However,  S.cerevisiae does not metabolize the sugar-phosphate so that glucosamine is toxic for S. cerevisiae. We have constructed a strain of S. cerevisiae that expresses the YlNAG1 gene from Y. lipolytica encoding the glucosamine-6-phosphate deaminase. This new strain utilizes glucosamine as carbon and nitrogen source. Metabolism of glucosamine in this new strain requires a functional respiratory chain.



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