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

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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

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Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Glycoproteins
Fungi
Proteins
Polysaccharides
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Glycoside Hydrolases
Secretory Pathway
Biosynthetic Pathways
Reducing Agents
Electrophoresis, Gel, Two-Dimensional
Mannose
Endoplasmic Reticulum
Methionine
Culture Media

Keywords

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

Cite this

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title = "Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei: Cellobiohydrolase I (CBHI) as a model protein",
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.",
keywords = "Glycosylation, Metabolic labelling, Protein transport, Two-dimensional gel electrophoresis",
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Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei : Cellobiohydrolase I (CBHI) as a model protein. / Pakula, Tiina M.; Uusitalo, Jaana; Saloheimo, Markku; Salonen, Katri; Aarts, Robert J.; Penttilä, Merja.

In: Microbiology, Vol. 146, No. 1, 01.01.2000, p. 223-232.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei

T2 - Cellobiohydrolase I (CBHI) as a model protein

AU - Pakula, Tiina M.

AU - Uusitalo, Jaana

AU - Saloheimo, Markku

AU - Salonen, Katri

AU - Aarts, Robert J.

AU - Penttilä, Merja

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N2 - 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.

AB - 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.

KW - Glycosylation

KW - Metabolic labelling

KW - Protein transport

KW - Two-dimensional gel electrophoresis

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