Effects of protein transport inhibitors on the distribution and secretion of the fusion protein RntA-EGFP in Aspergillus oryzae.
The distribution of the secreted protein ribonuclease T1 (RntA) fused with the enhanced green fluorescent protein (EGFP), RntA-EGFP, was visualized in hyphae of Aspergillus oryzae in the presence of a protein transport inhibitor, brefeldin A, cytochalasin A, or nocodazole. During treatment with the protein transport inhibitors, the distribution of RntA-EGFP changed and distinct patterns of fluorescence accumulation were observed. The addition of brefeldin A caused RntA-EGFP fluorescence to appear in reticular networks, and the disruption of the polymerization of actin filaments by cytochalasin A caused an increase in RntA-EGFP fluorescence intensity in the hyphae without accumulation in a specific cellular component. In contrast, RntA-EGFP fluorescence was distributed in different parts of a hypha during treatment with nocodazole, a compound that depolymerizes microtubules. In addition, quantitative analysis was performed using the RntA-EGFP visualization system to analyze the relative amount of RntA-EGFP secreted into the culture medium during treatment with the protein transport inhibitors.
Masai
Kumiko
K
Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Maruyama
Jun-Ichi
J
Nakajima
Harushi
H
Kitamoto
Katsuhiko
K
D001236:Aspergillus oryzae / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D020126:Brefeldin A / Q000494:pharmacology; D003572:Cytochalasins / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D025301:Hyphae / Q000502:physiology; D008856:Microscopy, Fluorescence; D008857:Microscopy, Interference; D008870:Microtubules / Q000502:physiology; D015739:Nocodazole / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006163:Ribonuclease T1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D003572:Cytochalasins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; C031218:cytochalasin A; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D006163:Ribonuclease T1; D015739:Nocodazole