Leaderless polypeptides efficiently extracted from whole cells by osmotic shock

Abstract

Three molecular foldases, DsbA, DsbC, and rotamase (ppiA), exhibited the unusual property of accumulating in an osmotically sensitive cellular compartment of Escherichia coli when their signal sequences were precisely removed by mutation. A mammalian protein, interleukin-1 (IL-1) receptor antagonist, behaved in a similar fashion in E. coli when its native signal sequence was deleted. These leaderless mutants (but not two control proteins overexpressed in the same system) were quantitatively extractable from whole cells by a variety of methods generally employed in the recovery of periplasmic proteins. A series of biochemical and genetic experiments showed that (i) leaderless DsbA (but not the wild type) was retained in a nonperiplasmic location; (ii) ␤-galactosidase fusions to leaderless DsbA (but not to the wild type) exhibited efficient ␣ complementation; (iii) none of the leaderless mutant proteins were substantially associated with cell membranes, even when they were overexpressed in cells; and (iv) leaderless DsbA was not transported to an osmotically sensitive compartment via a secA-or ftsZ-dependent mechanism. The observation that these proteins transit to some privileged cellular location by a previously undescribed mechanism(s)-absent their normal mode of (signal sequence-dependent) translocation-was unexpected. DsbA, rotamase, and IL-1, whose tertiary structures are known, appear to be structurally unrelated proteins. Despite a lack of obvious homologies, these proteins apparently have a common mechanism for intracellular localization. As this (putative) bacterial mechanism efficiently recognizes proteins of mammalian origin, it must be well conserved across evolutionary boundaries.Proteins destined for secretion to the periplasm or outer membrane of Escherichia coli typically contain a leader sequence at the amino terminus of the polypeptide chain. During translocation, this leader is generally cleaved from the mature protein (53, 67). The absence or inactivation of this leader by mutation results in a protein that cannot be efficiently translocated out of the cytoplasm by the cell's general secretory apparatus unless compensatory mutations are simultaneously introduced into one or more of the cell's secretory apparatus components (10,19,23,47).In addition to the well-documented translocation of proteins from cytoplasm to periplasm, selective protein localization to a variety of subcellular structures and organelles in E. coli has previously been documented (2,6,7,45,54,60); this has been observed even for proteins that are not native to cells (30,35,56). The mechanisms which mediate these localization events in bacterial cells remain to be fully elucidated.Proteins whose transport out of the cytoplasm is independent of the classical transmembrane secretory pathway in both prokaryotes (11,22,41,42) and eukaryotes (14,57,58,63,68) have previously been described. The protein interleukin-1␤ (IL-1␤), for example, is secreted from mammalian cells by a mechanism which is insensitive to brefeldin A, an inhib...

Molecular Sieve Mechanism of Selective Release of Cytoplasmic Proteins by Osmotically Shocked Escherichia coli

“…An interesting clue is provided by our finding that electroporation of cells, known to generate transient pores in biological membranes (19,24), causes selective release of the same subset of proteins, presumably passing through the same molecular sieve. Furthermore, solutions containing EDTA and a membrane-permeabilizing agent, such as Triton X-100 or polymyxin B, have been reported to extract from E. coli the same proteins as the osmotic shock procedure (23). It is tempting to speculate that the sudden swelling of the cytoplasm in osmotically shocked cells also leads to transient perforation of the plasma membrane.…”

“…The subset of released cytoplasmic proteins also includes thioredoxin (1,17), molecular chaperone DnaK (6,8), and proteins involved in the biosynthesis of enterobactin (9), among others (5). Some foreign proteins expressed in the cytoplasm of E. coli have also been successfully extracted by the osmotic shock procedure (16,23).…”

Investigation of Escherichia coli Dimethyl Sulfoxide Reductase Assembly and Processing in Strains Defective for the sec-Independent Protein Translocation System Membrane Targeting and Translocation

“…Procedures for restriction enzyme digestions, agarose gel electrophoresis, elution of DNA fragments from agarose gels, and separation of proteins using SDS-gel electrophoresis, phage P1 transduction, construction of deletion strains using the method of homologous recombination were carried out by standard procedures (25,26). Osmotic shock methods for preparing the periplasmic fraction and preparation of soluble and everted membrane vesicles by French press lysis were as described earlier (15,27). Protein was estimated by a modification of the Lowry procedure (28).…”

BfpU, a soluble protein essential for type IV pilus biogenesis in enteropathogenic Escherichia coli

“…However, we found that standard procedures such as osmotic shock and sphaeroplast formation led to an unacceptable degree of cell lysis. Therefore, a method using Triton X-100 was selected, as it proved to reliably separate periplasmic and cellular fractions (Thorstenson et al, 1997). The purity of each fraction was assessed using antibodies against the periplasmic maltose-binding protein and the cytoplasmic enzyme β-galactosidase.…”