The authors have developed methodology to study the kinetics of protein synthesis and secretion in filamentous fungi. Production of cellobiohydrolase I (CBHI) by Trichoderma reesei was studied by metabolic labelling of the proteins in vivo with [35S]methionine or [14C]mannose, and subsequent analysis of the labelled proteins using two-dimensional gel electrophoresis. Analysis of the different pI forms of the nascent proteins allowed monitoring of the maturation of CBHI during the transport along the biosynthetic pathway. The maturation of the pI pattern of CBHI as well as secretion into culture medium was prevented by treatment with the reducing agent DTT. The pI forms of CBHI detectable in the presence of DTT corresponded to the early endoplasmic reticulum forms of the protein. Removal of N-glycans by enzymic treatment (endoglycosidase H or peptide-N-glycosidase F), or chemical removal of both N- and O-glycans, changed the pI pattern of CBHI, showing that glycan structures are involved in formation of the different pI forms of the protein. By quantifying the labelled proteins during a time course, parameters describing protein synthesis and secretion were deduced. The mean synthesis time for CBHI under the conditions used was 4 min and the minimum secretion time was 11 min. The methodology developed in this study provides tools to reveal the rate-limiting factors in protein production and to obtain information on the intracellular events involved in the secretion process.
- Metabolic labelling
- Protein transport
- Two-dimensional gel electrophoresis