Glucoamylase::green fluorescent protein fusions to monitor protein secretion in Aspergillus niger.
A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA::sGFP by Western analysis. A strain containing the GLA::sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway.
Gordon
Caroline L
CL
School of Biological Sciences, Stopford Building, University of Manchester, Manchester M13 9PT, UK1.
Khalaj
Vahid
V
School of Biological Sciences, Stopford Building, University of Manchester, Manchester M13 9PT, UK1.
Ram
Arthur F J
AFJ
Centre for Phytotechnology, Institute for Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64, 2333 Al Leiden, The Netherlands2.
Archer
David B
DB
Department of Genetics and Microbiology, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK3.
Brookman
Jayne L
JL
School of Biological Sciences, Stopford Building, University of Manchester, Manchester M13 9PT, UK1.
Trinci
Anthony P J
APJ
School of Biological Sciences, Stopford Building, University of Manchester, Manchester M13 9PT, UK1.
Jeenes
David J
DJ
Department of Genetics and Microbiology, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK3.
Doonan
John H
JH
Department of Cell Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK5.
Wells
Brian
B
Department of Cell Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK5.
Punt
Peter J
PJ
TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, Utrechtseweg 48, PO Box 360, 3700 AJ Zeist, The Netherlands4.
van den Hondel
Cees A M J J
CAMJJ
TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, Utrechtseweg 48, PO Box 360, 3700 AJ Zeist, The Netherlands4.
Robson
Geoffrey D
GD
School of Biological Sciences, Stopford Building, University of Manchester, Manchester M13 9PT, UK1.
D001234:Aspergillus niger / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015153:Blotting, Western; D003470:Culture Media; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014170:Transformation, Genetic
D003470:Culture Media; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005087:Glucan 1,4-alpha-Glucosidase