SecB-mediated protein export need not occur via kinetic partitioning.
In Escherichia coli, the cytosolic chaperone SecB is responsible for the selective entry of a subset of precursor proteins into the Sec pathway. In vitro, SecB binds to a variety of unfolded substrates without apparent sequence specificity, but not native proteins. Selectivity has therefore been suggested to occur by kinetic partitioning of substrates between protein folding and SecB association. Evidence for kinetic partitioning is based on earlier observations that SecB blocks the refolding of the precursor form of maltose-binding protein (preMBP)(5) and slow-folding maltose-binding protein (MBP) mutants, but not faster-folding mature wild-type MBP. In order to quantitatively validate the kinetic partitioning model, we have independently measured each of the rate constants involved in the interaction of SecB with refolding preMBP (a physiological substrate of SecB) and mature MBP. The measured rate constants correctly predict substrate folding kinetics over a wide range of SecB, MBP, and preMBP concentrations. Analysis of the data reveals that, for many substrates, kinetic partitioning is unlikely to be responsible for SecB-mediated protein export. Instead, the ability of SecB-bound substrates to continue folding while bound to SecB and their ability to interact with other components of the secretory machinery such as SecA may be key opposing determinants that inhibit and promote protein export, respectively.
Krishnan
Beena
B
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
Kulothungan
S Rajendra
SR
Patra
Ashish K
AK
Udgaonkar
Jayant B
JB
Varadarajan
Raghavan
R
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D003198:Computer Simulation; D004926:Escherichia coli / Q000378:metabolism*; D005453:Fluorescence; D007700:Kinetics; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D014364:Tryptophan / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D018832:Molecular Chaperones; D050505:Mutant Proteins; D011498:Protein Precursors; C057842:SecB protein, Bacteria; C027132:1-anilino-8-naphthalenesulfonate; C038518:acrylodan; D014364:Tryptophan; D015081:2-Naphthylamine