6820a47d9415d4746382cc7bb1ad0ba1 pmc_34884915.pdf 2c2fda8c6f449de2a8e9ecc2d566af32efe8fe27 pmc_34884915.pdf 7ecb7b89373aab397c37b7a98e361f16a224ab54d4ee5649f45eca2c385c4644 pmc_34884915.pdf Title: The N-Acetylglucosamine Kinase from Yarrowia lipolytica Is a Moonlighting Protein Subject: In 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. Keywords: moonlighting; N-acetyglucosamine kinase; Yarrowia; yeast; N-acetyglucosaminetransporter Author: Carmen-Lisset Flores, Joaquín Ariño and Carlos Gancedo Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Dec 4 12:43:54 2021 CET ModDate: Sat Dec 4 12:47:52 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 8097052 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- UXXAKG+VnURWPalladioL Type 1 Custom yes yes no 10 0 LNFNTH+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 EQELPF+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 LGBYGK+URWPalladioL-BoldItal Type 1 Custom yes yes no 24 0 ZSBTUS+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 WLENKM+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 40 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 43 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 65 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 69 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 73 0 UWJUYN+CMSY10 Type 1 Builtin yes yes no 97 0 OEIBHX+PazoMath Type 1 Builtin yes yes no 123 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 157 0 CRJHCU+PazoMath-Italic Type 1 Builtin yes yes no 164 0 WABLSD+SFRM1000 Type 1 Custom yes yes no 172 0 BOLMEN+SFRM0900 Type 1 Custom yes yes no 180 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-22 02:29:54 CET RepresentationInformation: pmc_34884915.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-01-10 08:04:51 CET Size: 8097052 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 363 FreeObjects: 11 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Enzymatic Activity of Glucosamine-6-P Deaminase as Reporter for the Expression of the NAG Genes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Disruption of the Gene Encoding the NAGA Transporter Does Not Affect the Deregulated Expression of the NAG Genes in a Ylnag5 Mutant Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Assays to Evaluate the Enzymatic and Regulatory Activities of YlNag5 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Transcriptional Changes Caused by the Ylnag5 Mutation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: Strains and Culture Conditions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Plasmid Construction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Preparation of Extracts and Enzyme Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: -Galactosidase Plate Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Antibody Production Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Western Blot Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Random Mutagenesis of YlNAG5 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: RNA-Seq Experiments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Gene Ontology (GO) Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Info: Title: The N-Acetylglucosamine Kinase from Yarrowia lipolytica Is a Moonlighting Protein Author: Carmen-Lisset Flores, Joaquín Ariño and Carlos Gancedo Subject: In 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. 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