Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei: Cellobiohydrolase I (CBHI) as a model protein

    Research output: Contribution to journalArticleScientificpeer-review

    40 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)223-232
    Number of pages10
    JournalMicrobiology
    Volume146
    Issue number1
    DOIs
    Publication statusPublished - 1 Jan 2000
    MoE publication typeA1 Journal article-refereed

    Keywords

    • Glycosylation
    • Metabolic labelling
    • Protein transport
    • Two-dimensional gel electrophoresis

    Fingerprint

    Dive into the research topics of 'Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei: Cellobiohydrolase I (CBHI) as a model protein'. Together they form a unique fingerprint.

    Cite this