Abstract
Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphatases / metabolism
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Bacterial Proteins / metabolism
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Carrier Proteins / chemistry
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Carrier Proteins / isolation & purification
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Carrier Proteins / metabolism
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Endopeptidase K / metabolism
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Escherichia coli / metabolism*
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / isolation & purification
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Escherichia coli Proteins / metabolism*
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Kinetics
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Membrane Transport Proteins / metabolism
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Mutagenesis
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Mutant Proteins / chemistry*
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Mutant Proteins / isolation & purification
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Mutant Proteins / metabolism*
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Mutation / genetics*
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Periplasmic Binding Proteins / chemistry*
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Periplasmic Binding Proteins / isolation & purification
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Periplasmic Binding Proteins / metabolism*
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Protein Folding*
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Protein Precursors / chemistry
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Protein Precursors / isolation & purification
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Protein Precursors / metabolism
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Protein Transport
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SEC Translocation Channels
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SecA Proteins
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Spectrometry, Fluorescence
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Thermodynamics
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Tryptophan
Substances
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Bacterial Proteins
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Carrier Proteins
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Escherichia coli Proteins
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MalE protein, E coli
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Membrane Transport Proteins
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Mutant Proteins
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Periplasmic Binding Proteins
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Protein Precursors
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SEC Translocation Channels
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preMBP protein, E coli
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Tryptophan
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Endopeptidase K
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Adenosine Triphosphatases
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SecA Proteins