title: Functional YFP-tagging of the essential GDP-mannose transporter reveals an important role for the secretion related small GTPase SrgC protein in maintenance of Golgi bodies in Aspergillus niger.
abstract: The addition of mannose residues to glycoproteins and glycolipids in the Golgi is carried out by mannosyltransferases. Their activity depends on the presence of GDP-mannose in the lumen of the Golgi. The transport of GDP-mannose (mannosyl donor) into the Golgi requires a specific nucleotide sugar transport present in the Golgi membrane. Here, we report the identification and functional characterization of the putative GDP-mannose transporter in Aspergillus niger, encoded by the gmtA gene (An17g02140). The single GDP-mannose transporter was identified in the A. niger genome and deletion analysis showed that gmtA is an essential gene. The lethal phenotype of the gmtA could be fully complemented by expressing an YFP-GmtA fusion protein from the endogenous gmtA promoter. Fluorescence studies revealed that, as in other fungal species, GmtA localized as punctate dots throughout the hyphal cytoplasm, representing Golgi bodies or Golgi equivalents. SrgC encodes a member of the Rab6/Ypt6 subfamily of secretion-related GTPases and is predicted to be required for the Golgi to vacuole transport. Loss of function of the srgC gene in A. niger resulted in strongly reduced growth and the inability to form conidiospores at 37°C and higher. Furthermore, the srgC disruption in the A. niger strain expressing the functional YFP-GmtA fusion protein led to an apparent 'disappearance' of the Golgi-like structures. The analysis suggests that SrgC has an important role in maintaining the integrity of Golgi-like structures in A. niger.
authors.affiliation: Department Molecular Microbiology and Biotechnology, Institute of Biology, Leiden University, Sylviusweg 72, Leiden, The Netherlands.
authors.forename: Neuza D S P
authors.initials: ND
authors.lastname: Carvalho
authors.forename: Mark
authors.initials: M
authors.lastname: Arentshorst
authors.forename: Xavier O
authors.initials: XO
authors.lastname: Weenink
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Punt
authors.forename: Cees A M J J
authors.initials: CA
authors.lastname: van den Hondel
authors.forename: Arthur F J
authors.initials: AF
authors.lastname: Ram
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D020043:Genes, Essential; D005800:Genes, Fungal; D006056:Golgi Apparatus / Q000378:metabolism*; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D005656:Fungal Proteins; C428326:GDP-mannose transporter; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D006155:Guanosine Diphosphate Mannose; D020559:Monomeric GTP-Binding Proteins