The major cellulases CBH‐1 and CBH‐2 ofNeurospora crassarely on distinct ER cargo adaptors for efficient ER‐exit

Abstract

Filamentous fungi are native secretors of lignocellulolytic enzymes and are used as protein-producing factories in the industrial biotechnology sector. Despite the importance of these organisms in industry, relatively little is known about the filamentous fungal secretory pathway or how it might be manipulated for improved protein production. Here, we use Neurospora crassa as a model filamentous fungus to interrogate the requirements for trafficking of cellulase enzymes from the endoplasmic reticulum to the Golgi. We characterized the localization and interaction properties of the p24 and ERV-29 cargo adaptors, as well as their role in cellulase enzyme trafficking. We find that the two most abundantly secreted cellulases, CBH-1 and CBH-2, depend on distinct ER cargo adaptors for efficient exit from the ER. CBH-1 depends on the p24 proteins, whereas CBH-2 depends on the N. crassa homolog of yeast Erv29p. This study provides a first step in characterizing distinct trafficking pathways of lignocellulolytic enzymes in filamentous fungi.

Heterotrimeric G-Protein Signaling Is Required for Cellulose Degradation in Neurospora crassa

“…Previous work has determined the transcription factors responsible for cellulase induction (10,34), the intracellular molecule that induces cellulolytic enzymes (54), and the adaptor proteins needed for efficient exit of cellulases from the endoplasmic reticulum (ER) to the Golgi apparatus in fungi (48). Our results contribute to this body of knowledge by providing a comprehensive analysis illustrating the importance of G-protein signaling during upstream regulation of cellulose sensing and cellulase induction.…”

“…Since the proteins appear to be made in these mutant backgrounds (Fig. 1 and S3), these may affect posttranslational modifications required for the cellulase enzymes to be functional (47,48). It should be noted that since the time point selected for our study was early during cellulase induction, it is possible that levels of these mRNAs may be higher at later times after transfer to Avicel.…”

“…Cellulase mRNA levels in the Δcpc-2 mutant are either similar to or greater than those in the wild type, but the Δcpc-2 mutant lacks detectable cellulase activity and does not respond to cAMP. We propose that CPC-2 may impact posttranslational modifications of cellulases required for activity, perhaps through translational control of the modifying proteins (47,48). Additional experiments are necessary to determine the exact posttranscriptional effect of CPC-2 on cellulase production in N. crassa.…”

Genetic dissection of the secretory route followed by a fungal extracellular glycosyl hydrolase

“…Therefore the fact that CG forms are markedly abundant in InuA extracted from mycelia strongly suggests that, at least for this cargo, ER exit is limiting for secretion in A. nidulans . In this particular regard we note that a recent study demonstrated that CBH‐1 and CBH‐2, the major cellulases of Neurospora crassa , use different cargo adaptors (the p24 complex and Erv29 respectively) to the COPII coat during ER exit (Starr et al, ). Future studies will address whether one or more A. nidulans cargo adaptors mediating incorporation of InuA into COPII carriers is/are limiting.…”

Off the wall: The rhyme and reason of Neurospora crassa hyphal morphogenesis

“…This modification is necessary for Chs3p to achieve a competent conformation required for its ER exit ( Lam et al, 2006 ). Moreover, the ER export cargo Chs7p ( Starr et al, 2018 ) is responsible for directing the appropriate folding of Chs3p, avoiding its aggregation in the ER ( Kota and Ljungdahl, 2005 , Trilla et al, 1999 ). Chs3p is then transported to Golgi.…”