Abstract
It has been proposed (Randall, L. L., and Hardy, S. J. S. (1986) Cell 46, 921-928) that export of protein involves a kinetic partitioning between the pathway that leads to productive export and the pathway that leads to the folding of polypeptides into a stable conformation that is incompatible with export. As predicted from this model, a decrease in the rate of export of maltose-binding protein to the periplasmic space in Escherichia coli resulting from a defect in the leader sequence was able to be partially overcome by a mutation that slowed the folding of the precursor, thereby increasing the time in which the polypeptide was competent for export. (Liu, G., Topping, T. B., Cover, W. H., and Randall, L. L. (1988) J. Biol. Chem. 263, 14790-14793). Here we describe mutations of the gene encoding ribose-binding protein that were selected as suppressors of a defect in export of that protein and that alter the folding pathway. We propose that selection of such suppressors may provide a general method to obtain mutations that affect the folding properties of any protein that can be expressed and exported in E. coli.
Highlights
If enoughaminoacyl involves a kinetic partitioning between the pathway that leads to productive export and the pathway that leads to the folding of polypeptides into a stable conformation that is incompatible with export
As predicted from this model, a decrease in the rate of export of maltose-binding protein to the periplasmic space in Escherichia coli resulting from a defect in the leader sequence was able to be partially overcome by a mutation that slowed the folding of the precursor, thereby increasing the time in which the polypeptide was competent for export
This method may be of general application to that selection of such suppressors may provide a gen- studies of proteins that can be exported by Escherichia coli
Summary
Materials-Guanidinium chloride (ultrapure) was purchased from Schwartz/Mann. HEPES' and Trizma were purchased from Sigma. Isolation of Reuertants-The strain A1287 (rbsB::TnlO) harboring a plasmid that carries rbsBIO3, which encodes a precursor of ribosebinding protein with a defective leader sequence, does not export ribose-binding protein and grows poorly on minimal salts medium supplemented with 0.05% ribose. To determine if the suppressor mutations were true revertants or were mutations at a second site in rbsB103, we determined if the BstNI restriction site, present in the wild-type leader sequencebut abolished by the mutational change in rbsB103, had been restored (Iida et al, 1985).The plasmids carrying the suppressor mutations, rbsB2Ol and rbsB202, described in this study were named pH07 and pTS6, respectively. The large fragment (5.0 kb) carries all the vector and the first 30 codons of the rbsB gene whichencode the leader sequence and the amino terminus of ribose-binding protein. CP627 CP628 CP629 CP630 CP631 CP632 CP633 CP649 CP650 HB786 HB787 HB1151 HB1152 IQ85 MC4100
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