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 prL4 (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prL4666, 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 prUi 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 prUA666. 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.Proteins exported across the cytoplasmic membrane of Escherichia coli cells are usually synthesized with a cleavable, amino-terminal signal peptide, which is thought to be chiefly responsible for initiating the export process. This structure includes a central hydrophobic core followed by a somewhat less apolar signal peptidase processing site and preceded by a hydrophilic segment that exhibits a net positive charge due to the presence of one to three basic residues (39). The early mature region of such proteins generally lacks a net positive charge (40). For integral cytoplasmic membrane proteins, a region of net positive charge is usually found immediately adjacent to one end of each membrane-spanning stretch of hydrophobic amino acids (41). In both cases, the transmembrane orientation of the hydrophobic domains is thought to be primarily determined by the distribution of basic residues at either end, with the most positively charged end residing in the cytoplasm. This "positive-inside rule" was initially proposed by von Heijne (41).In support of the positive-inside rule, several studies have shown that systematically reducing the net charge of the signal peptide hydr...

The efficient export of the Escherichia coli maltose-binding protein (MBP) is known to be SecB dependent, whereas ribose-binding protein (RBP) export is SecB independent. When the MBP and RBP signal peptides were exchanged precisely at the signal peptidase processing sites, the resultant RBP-MBP and MBP-RBP hybrid proteins both were efficiently exported in SecB+ cells. However, only MBP-RBP was efficiently exported in SecB-cells; RBP-MBP exhibited a significant export defect, a finding that was consistent with previous proposals that SecB specifically interacts with the mature moiety of precursor MBP to promote export. The relatively slow, totally posttranslational export mode exhibited by certain mutant RBP and MBP-RBP species in SecB+ cells was not affected by the loss of SecB. In contrast, MBP and RBP-MBP species with similarly altered signal peptides were totally export defective in SecB-cells. Both export-defective MBP and RBP-MBP interfered with SecB-mediated protein export by depleting cells of functional SecB. In contrast, neither export-defective RBP nor MBP-RBP elicited such an interference effect. These and other data indicated that SecB is unable to interact with precursor RBP or that any interaction between these two proteins is considerably weaker than that of SecB with precursor MBP. In addition, no correlation could be established between a SecB requirement for export and PrlA-mediated suppression of signal peptide export defects. Finally, previous studies have established that wild-type MBP export can be accomplished cotranslationally, whereas wild-type RBP export is strictly a posttranslational process. In this study, cotranslational export was not detected for either MBP-RBP or RBP-MBP. This indicates that the export mode exhibited by a given precursor protein (cotranslational versus posttranslational) is determined by properties of both the signal peptide and the mature moiety.

The pathogenic Neisseria, N. gonorrhoeae and N. meningitidis, possess an outer membrane protein (OMP), designated H.8, with a conserved monoclonal antibody (MAb)-binding epitope. We determined the DNA sequence of a gonococcal H.8 gene, and confirmed the relationship between the cloned gene and the H.8 OMP by constructing a gonococcal mutant lacking H.8. The predicted H.8 OMP is a lipoprotein 71 amino acids in length, composed of 13 repeats of a consensus sequence AAEAP with perfect 5-residue periodicity. The AAEAP units form a repeating epitope that comprises the entire predicted sequence of the protein.

SecB, a molecular chaperone involved in protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative. The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB. SecB has no affinity for native, stably folded polypeptides but interacts tightly with polypeptides that are nonnative. In order to better understand the nature of the binding, we have examined the interaction of SecB with intermediates along the folding pathway of maltose-binding protein. Taking advantage of forms of maltosebinding protein that are altered in their folding properties, we show that the first intermediate in folding, represented by the collapsed state, binds to SecB, and that the polypeptide remains active as a ligand until it crosses the final energy barrier to attain the native state.

Cytochromes P450 2B metabolize a variety of compounds and have provided an excellent framework for identifying determinants of substrate specificity. Among the rat 2B enzymes, a puzzling difference has emerged between the reported substrate specificity of purified hepatic 2B2 and that of certain 2B1 mutants containing 2B1 --> 2B2 substitutions. To address these discrepancies, we have characterized two 2B2 variants. A cDNA clone designated 2B2FF was obtained from phenobarbital-induced Lewis rats and, like some previously isolated variants, was found to contain phenylalanine at positions 58 and 114. A second 2B2 clone was generated by restoring Leu and Ile, respectively, at these positions. These enzymes were expressed in Escherichia coli and analyzed with androstenedione, testosterone, progesterone, ethoxycoumarin, benzyloxyresorufin, and pentoxyresorufin. The expressed 2B2 variants metabolized most substrates at rates that were 1-9% of those of 2B1. When steroid regio- and stereospecificity was examined, the metabolite profiles of expressed 2B2 and 2B2FF conflicted with the 16beta- and 16alpha-hydroxylation observed for purified hepatic 2B2 from Sprague-Dawley rats. These and other results suggested that the purified hepatic 2B2 contained a small percent of the 2B1 enzyme. Masses of tryptic peptides were consistent with identity between purified hepatic 2B2 and 2B2FF. On the basis of a three-dimensional homology model and the construction and analysis of 2B2 mutants, residues 114, 363, 367, and 478 were identified as determinants of substrate specificity. In addition, 2B1 and the expressed 2B2 variants showed differential susceptibility to the mechanism-based inactivators chloramphenicol and N-(2-p-nitrophenethyl)chlorofluoroacetamide.