Post-translational export of maltose-binding protein in Escherichia coli strains harboring malE signal sequence mutations and either prl+ or prl suppressor alleles.

J P Ryan,P J Bassford

doi:10.1016/s0021-9258(17)38647-7

Abstract

We have studied the export kinetics of the maltose-binding protein (MBP) of Escherichia coli, the malE gene product, when it is synthesized with either a wildtype signal sequence or with a mutationally altered signal sequence that affects the efficiency of secretion to the periplasm. Our results confirm a very rapid export process for the wild-type protein and, in contrast, reveal a relatively slow post-translational mode of export for the altered precursor species. For each different signal sequence mutant, a fraction of the precursor MBP pool that is proportional to the strength of the export defect appears to never exit the cytoplasm. We have also analyzed MBP export in strains harboring prl mutations that suppress malE signal sequence mutations and are thought to somehow alter the specificity of the cell's protein export machinery. The introduction of different prl alleles has no apparent effect on wild-type MBP export but increases both the amount of mutant MBP that is exported and the rate at which this is accomplished. In fact, the presence of two different prl alleles in the same strain can act synergistically in suppressing MBP export defects. The inhibition of total protein synthesis with chloramphenicol can also increase the proportion of pMBP that is post-translationally exported in these strains. A model that describes the initial steps in MBP export is presented.

Highlights

W e have studied the exportkinetics of the maltose- Certain aspects of the export of the periplasmic MBP’ of binding protein (MBP) of Escherichia coli, the m d E Escherichia coli, the malE product, are consistent with the gene product, when it is synthesized with either a wild- eukaryotic model as it has been applied to prokaryotic cells type signal sequence or with a mutationally altered (4).The MBP isinitially synthesized as aprecursor having a signal sequence that affects theefficiency of secretion 26-residue cleavable signal peptide that is similar to those to the periplasm
The inhibition of total protein synthesis wicthloramphenicol can increase the proportion of pMBP that is post-translationally exported in these strainAs.model that describes the initial steps in MBP export is presented
Much of the information concerning the mechanism of MBP synthesis and secretion has resulted from the study of mutants affecting these processes.Strains harboring five different malE point mutationsthat altertheMBP signal peptide accumulate export-defective pMBP in the cytoplasm (Refs. 9-11; see Fig. 1).Extragenic suppressor mutations have been obtained in at least four different loci that restore, to varying extents, export of MBP and other proteins having defective signal peptides (11-14)

Summary

Introduction

W e have studied the exportkinetics of the maltose- Certain aspects of the export of the periplasmic MBP’ of binding protein (MBP) of Escherichia coli, the m d E Escherichia coli, the malE product, are consistent with the gene product, when it is synthesized with either a wild- eukaryotic model as it has been applied to prokaryotic cells type signal sequence or with a mutationally altered (4).The MBP isinitially synthesized as aprecursor having a signal sequence that affects theefficiency of secretion 26-residue cleavable signal peptide that is similar to those to the periplasm. Cell Fractionation and Proteinase K Accessibility Experiments- a good indicator of MBP export from the cytoplasm.

Results

Conclusion