A Study of the Protein Secretory Pathway of Aspergillus niger Using a Glucoamylase–GFP Fusion Protein

• Published in: Fungal genetics and biology Vol. 32; no. 1; pp. 55 - 65
• Main Authors: Khalaj, Vahid, Brookman, Jayne L, Robson, Geoffrey D
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2001
• Subjects: actin; Aspergillus niger; Aspergillus niger - drug effects; Aspergillus niger - genetics; Aspergillus niger - metabolism; brefeldin A; Brefeldin A - pharmacology; Cold Temperature; Culture Media; cytochalasin; cytochalasin A; Cytochalasins - pharmacology; Endoplasmic Reticulum - secretion; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Fungal Proteins - secretion; GFP; GFP protein; Glucan 1,4-alpha-Glucosidase - genetics; Glucan 1,4-alpha-Glucosidase - isolation & purification; Glucan 1,4-alpha-Glucosidase - secretion; glucoamylase; Glucose - metabolism; golgi; Golgi Apparatus - secretion; Green Fluorescent Proteins; Luminescent Proteins; Microscopy, Fluorescence; microtubule; Microtubules; nocodazole; Nocodazole - pharmacology; protein secretion; Protein Synthesis Inhibitors - pharmacology; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - secretion; Vacuoles - metabolism; GFP; microtubule; actin; Aspergillus niger; brefeldin A; cytochalasin; golgi; ER; protein secretion; glucoamylase; nocodazole
• ISSN: 1087-1845; 1096-0937
• DOI: 10.1006/fgbi.2000.1245

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.