A study of the protein secretory pathway of Aspergillus niger using a glucoamylase-GFP fusion protein.
The effect of various treatments that block protein secretion was visualized in Aspergillus niger using a strain expressing a glucoamylase-GFP fusion protein. Cold shock caused the retention of the fusion protein in a reticulate network (ER) with brighter nodes that may represent Golgi bodies. Treatment of germlings with brefeldin A (BFA) also initially caused accumulation within the ER but prolonged exposure led to the formation and targeting of the fusion protein to vacuoles from the ER. Disruption of actin with cytochalasin A initially led to a faint diffuse accumulation and ultimately to the formation of aggregated bodies which were not vacuoles, suggesting that the actin cytoskeleton is important in secretory vesicle transport. Disruption of microtubules with nocodazole led to hyperbranching but did not cause intracellular accumulation, suggesting that microtubules play a role in directing vesicle transport rather than vesicle movement per se. Treatment of regenerating protoplasts confirmed that BFA and cytochalasin but not nocodazole inhibited protein secretion. When germlings were subjected to carbon starvation, vacuolation was rapidly initiated throughout the hyphae and GFP fluorescence was visible in some of the vacuoles, indicating retargeting of the fusion protein from the secretory pathway to the vacuoles.
Khalaj
V
V
School of Biological Sciences, University of Manchester, 1.800 Stopford Building, Manchester, M13 9PT, United Kingdom.
Brookman
J L
JL
Robson
G D
GD
D001234:Aspergillus niger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D003080:Cold Temperature; D003470:Culture Media; D003572:Cytochalasins / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D008870:Microtubules; D015739:Nocodazole / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
D003470:Culture Media; D003572:Cytochalasins; D005656:Fungal Proteins; D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; C031218:cytochalasin A; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D005087:Glucan 1,4-alpha-Glucosidase; D005947:Glucose; D015739:Nocodazole