The Effects of Drugs Inhibiting Protein Secretion in the Filamentous Fungus Trichoderma reesei

Abstract

To study the mechanisms of protein secretion as well as the cellular responses to impaired protein folding and transport in filamentous fungi, we have analyzed Trichoderma reesei cultures treated with chemical agents that interfere with these processes, dithiothreitol, brefeldin A, and the Ca 2؉ -ionophore A23187. The effects of the drugs on the kinetics of protein synthesis and transport were characterized using metabolic labeling of synthesized proteins. Cellobiohydrolase I (CBHI, Cel7A), the major secreted cellulase, was analyzed as a model protein. Northern analysis showed that under conditions where protein transport was inhibited (treatments with dithiothreitol or brefeldin A) the unfolded protein response pathway was activated. The active form of the hac1 mRNA that mediates unfolded protein response signaling was induced, followed by induction of the foldase and chaperone genes pdi1 and bip1. Concomitant with the activation of the unfolded protein response pathway, the transcript levels of genes encoding secreted proteins, like cellulases and xylanases, were drastically decreased, suggesting a novel type of feedback mechanism activated in response to impairment in protein folding or transport (repression under secretion stress (RESS)). By studying expression of the reporter gene lacZ under cbh1 promoters of different length, it was shown that the feedback response was mediated through the cellulase promoter.

Increased Production of Xylanase by Expression of a Truncated Version of the xyn11A Gene from Nonomuraea flexuosa in Trichoderma reesei

“…T. reesei has been described as responding to secretion stress by decreasing the level of transcription from genes that encode the major extracellular proteins (e.g., the cellulase genes). This phenomenon has been named as a "RESS response" (repression under secretion stress) (23). We did not study the expression of the cellulase genes by Northern blotting.…”

Deciphering the Effect of the Different N-Glycosylation Sites on the Secretion, Activity, and Stability of Cellobiohydrolase I from Trichoderma reesei

“…The ability of Asn-linked glycans to extrinsically enhance protein folding and reduce aggregation in vivo by directing the protein into the calnexin and/or calreticulin (CNX/CRT) folding cycle has been well established (4). If proper folding cannot be achieved, unfolded protein response (UPR) occurs or glycoproteins are marked for ER-associated degradation, which is also directed by the glycan tag (5)(6)(7)(8). N-glycans can also exert effects on protein stabilization and folding acceleration through an apparent intrinsic chemical mechanism (9)(10)(11)(12)(13).…”

Effect of manganese on the secretion of manganese-peroxidase by the basidiomycete Ceriporiopsis subvermispora

“…First, the effectiveness of cycloheximide (CHX) as inhibitor of protein synthesis and the protocol previously established for that purpose (Pakula et al, 2003) was determined as follows: cultures were grown for 10 days in the absence of manganese and then supplemented with CHX (250 lg/ml). Ten minutes later, 35 S-methionine (0.5 mCi, New England Nuclear) was added to the cultures during an additional period of 50 min.…”