LEADERLESS POLYPEPTIDES EFFICIENTLY EXTRACTED FROM WHOLE CELLS BY OSMOTIC SHOCK

Three molecular foldases, DsbA, DsbC, and rotamase (ppiA), exhibited t he unusual property of accumulating in an osmotically sensitive cellul ar compartment of Escherichia coli when their signal sequences Mere pr ecisely 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 quantitat ively extractable from,whole cells by a variety of methods generally e mployed in the recovery of periplasmic proteins, A series of biochemic al and genetic experiments showed that (i) leaderless DsbA (but not th e wild type) was retained in a nonperiplasmic location; (ii) beta-gala ctosidase fusions to leaderless DsbA (but not to the wild type) exhibi ted efficient cw 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 tra nsported to an osmotically sensitive compartment via a secA- or ftsZ-d ependent mechanism. The observation that these proteins transit to som e privileged cellular location by a previously undescribed mechanism(s )-absent their normal mode of (signal sequence-dependent) translocatio n-was unexpected. DsbA, rotamase, and IL-1, whose tertiary structures are known, appear to be structurally unrelated proteins, Despite a lac k of obvious homologies, these proteins apparently have a common mecha nism for intracellular localization, As this (putative) bacterial mech anism efficiently recognizes proteins of mammalian origin, it must be well conserved across evolutionary boundaries.