In vivo imaging of endoplasmic reticulum and distribution of mutant α-amylase in Aspergillus oryzae.
Properly folded proteins destined for secretion exit through a specific subdomain of the endoplasmic reticulum (ER) known as transitional ER (tER) sites or ER exit sites (ERES). While such proteins in filamentous fungi localize at the hyphal tips overlapping the Spitzenkörper, the distribution of misfolded proteins remains unknown. In the present study, we analyzed the distribution of mutant protein as well as ER and tER sites visualized by expression of AoClxA and AoSec13 fused with fluorescent protein, respectively, in the filamentous fungus Aspergillus oryzae. Discrete tER subdomains were visualized as the punctate dots of AoSec13 overlapping or associated with AoClxA distribution. Both ER and tER sites were concentrated near hyphal tips and formed apical gradients. Interestingly, while the expression of wild-type α-amylase fusion protein (AmyB-mDsRed) showed its localization coinciding with the Spitzenkörper, a disulfide bond-deletion in AmyB causing its misfolding resulted in its accumulation in the subapical and basal ER, creating a reciprocal gradient to the tER sites. Furthermore, the reciprocal gradient enabled a clear distinction between the tER sites and the mutant AmyB accumulation sites near the apex. Based on these findings, we conclude that A. oryzae accumulates aberrant proteins toward basal hyphae while maintaining polarized tER sites for secretion of properly folded proteins at the hyphal tip.
Kimura
Shinya
S
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan.
Maruyama
Jun-ichi
J
Watanabe
Taisuke
T
Ito
Yukishige
Y
Arioka
Manabu
M
Kitamoto
Katsuhiko
K
D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D025301:Hyphae / Q000201:enzymology / Q000235:genetics; D009154:Mutation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics* / Q000378:metabolism*
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000516:alpha-Amylases