Effect of charge distribution on the translocation of an inhomogeneously charged polymer through a nanopore

We investigate the voltage-driven translocation of an inhomogeneously charged polymer through a nanopore by utilizing discrete and continuous stochastic models. As a simplified illustration of the effect of charge distribution on translocation, we consider the translocation of a polymer with a single charged site in the presence and absence of interactions between the charge and the pore. We find that the position of the charge that minimizes the translocation time in the absence of pore--polymer interactions is determined by the entropic cost of translocation, with the optimum charge position being at the midpoint of the chain for a rodlike polymer and close to the leading chain end for an ideal chain. The presence of attractive or repulsive pore--charge interactions yields a shift in the optimum charge position towards the trailing end and the leading end of the chain, respectively. Moreover, our results show that strong attractive or repulsive interactions between the charge and the pore lengthen the translocation time relative to translocation through an inert pore. We generalize our results to accommodate the presence of multiple charged sites on the polymer. Our results provide insight into the effect of charge inhomogeneity on protein translocation through biological membranes.Comment: Submitted to Journal of Chemical Physic

A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

The prA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several priA mutants. The export efficiency can be substantial; in a strain carrying the prUA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all

Analysis of mutational alterations in the hydrophilic segment of the maltose-binding protein signal peptide.

Oligonucleotide-directed mutagenesis was employed to investigate the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the protein export process. The three basic residues residing at the amino terminus of the signal peptide were systematically substituted with neutral or acidic residues, decreasing the net charge in a stepwise fashion from +3 to -3. It was found that a net positive charge was not absolutely required for MBP export to the periplasm. However, export was most rapid and efficient when the signal peptide retained at least a single basic residue and a net charge of +1. The nature of the adjacent hydrophobic core helped to determine the effect of charge changes in the hydrophilic segment on MBP export, which suggested that these two regions of the signal peptide do not have totally distinct functions. Although the stepwise decrease in net charge of the signal peptide also resulted in a progressive decrease in the level of MBP synthesis, the data do not readily support a model in which MBP synthesis and export are obligately coupled events. The export defect resulting from alterations in the hydrophilic segment was partially suppressed in strains harboring certain prl alleles but not in strains harboring prlA alleles that are highly efficient suppressors of signal sequence mutations that alter the hydrophobic core

Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP.

Mutations that reduce the net positive charge within the hydrophilic segments of the signal peptides of several prokaryotic exported proteins can result in a reduction in the rate of protein export, as well as a reduction in protein synthesis (M. N. Hall, J. Gabay, and M. Shwartz, EMBO J. 2:15-19, 1983; S. Inouye, X. Soberon, T. Franceschini, K. Nakamura, K. Itakura, and M. Inouye, Proc. Natl. Acad. Sci. USA 79:3438-3441, 1982; J. W. Puziss, J. D. Fikes, and P. J. Bassford, Jr., J. Bacteriol. 171:2302-2311, 1989). This result has been interpreted as evidence that the hydrophilic segment is part of a mechanism that obligatorily couples translation to protein export. We have investigated the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the export and synthesis of MBP. Deletion of the entire hydrophilic segment from the MBP signal peptide resulted in a defect in MBP export, as well as a dramatic reduction in total MBP synthesis. Suppressor mutations that lie upstream of the malE coding region were isolated. These mutations do not affect MBP export but instead were shown to partially restore MBP synthesis by increasing the efficiency of MBP translational initiation. In addition, analysis of a series of substitution mutations in the second codon of certain malE alleles demonstrated that MBP export and synthesis can be independently affected by mutations in the hydrophilic segment. Finally, analysis of alterations in the hydrophilic segment of the ribose-binding protein signal peptide fused to the mature moiety of the MBP has revealed that the role of the hydrophilic segment in the export process can be functionally separated from any role in translation. Taken together, these results strongly suggest that the hydrophilic segment of the MBP signal peptide is not involved in a mechanism that couples MBP translation to export and argue against the presence of a mechanism that obligatorily couples translation to protein export in Escherichia coli

Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations.

It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process. The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4. This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0. One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective. Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects. Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E. coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide. In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666. These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane