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 Spitzenkorper the distribution of misfolded proteins remains unknown In the present study we analyzed the distnbution 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 a-amylase fusion protein (AmyB-mDsRed) showed its localization coinciding with the Spitzenkorper 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 (C) 2010 Elsevier Inc All rights reserved