The Sec-dependent protein export pathway of Escherichia coli is responsible for translocation of secretory proteins across the inner membrane to final destinations in the periplasm or outer membrane. Secretory proteins, also called preproteins, are synthesized with a cleavable amino terminal signal sequence that functions both to slow folding of the preprotein and to aid in recognition of the secretory protein by export factors. Export of many, but not all, secretory proteins is dependent on interaction with SecB, a cytoplasmic chaperone that maintains the preprotein in a loosely folded conformation competent for translocation. Both SecB and the preprotein provide binding sites for SecA, a peripheral membrane ATPase. SecA targets the preprotein to the membranous translocase complex composed of SecY, SecE, SecG, SecD, SecF, and YajC. Formation of the complete translocase complex promotes an ATP binding and hydrolysis cycle by SecA that results in segmental translocation of the secretory protein across the membrane [1–4].
The signal sequence is crucial for efficient translocation; mutations in the signal sequence significantly reduce export of the preprotein and complete deletions of the signal sequence eliminate essentially all export [5–8]. Selection for extragenic suppressors of such export defective preproteins led to the identification of the prl alleles of secY (prlA), secE (prlG), secA (prlD), and more recently secG (prlH) [6, 9–11]. Early hypotheses predicted that the Prl suppressors function by expanded or altered interactions with signal sequences, facilitating recognition of mutant as well as wild type signal sequences [6].
More recent observations support an alternative mechanism of action for the PrlA/SecY and PrlG/SecE suppressors [8, 12]. First, PrlA/G-mediated suppression does not exhibit allele specificity; all prlA and all prlG alleles that have been examined suppress all signal sequence mutations, implying that suppression is not due to a specific altered interaction that allows recognition of a mutant signal sequence [8, 12]. Furthermore, all of the prlA and prlG alleles suppress complete deletions of the signal sequence, suggesting that an interaction between the signal sequence and the Prl suppressor is not necessary for suppression or even for export [5, 8, 13]. Based on these observations, it was proposed that the PrlA and PrlG suppressors do not function through altered interactions with signal sequences, but rather by loss of recognition [8, 12]. The wild type SecE and SecY proteins are thought to function in concert to proofread the signal sequence of secretory proteins, rejecting defective precursors from the export pathway. The PrlA (SecY) and PrlG (SecE) suppressors are compromised in their ability to proofread, allowing export of proteins with mutations, or even complete deletions, of the signal sequence.
This proofreading model predicts that the critical step for translocation of a signal sequence-defective secretory protein is recognition by SecB and subsequent presentation to SecE/Y. SecB binds portions of the mature secretory protein [14–18], and SecA would bind to the SecB-precursor complex by virtue of its ability to bind SecB [19, 20] as well as the preprotein [21–23]. Thus, SecA-mediated targeting of the preprotein to translocase would occur. In a wild type cell, the mutant precursor would be rejected at this point by SecY/E, but in a PrlA or PrlG suppressor strain, rejection would not occur and the mutant secretory protein would be exported.
In fact, all of the PrlA and PrlG suppressors are completely dependent on functional SecB for manifestation of suppressor activity [5, 8]. Even certain proteins that are not normally SecB-dependent for translocation become SecB-dependent when they contain a signal sequence mutation and are exported via the suppressor pathway. For example, PhoA is normally exported independent of SecB. Deletion of the signal sequence from phoA severely compromises export; export is restored in a prlA suppressor strain. However, this suppression is dependent on SecB [5]. Therefore, there are characteristics of the mature portion of PhoA that promote recognition by SecB, and this recognition is essential for PrlA-mediated suppression.
Although prlA and prlG strains export secretory proteins that completely lack a signal sequence, there is no evidence of export of any cytoplasmic proteins in a prl suppressor strain [13]. That is, proteins that are supposed to remain in the cytoplasm are not mislocalized to the periplasm. It has been suggested that perhaps cytoplasmic proteins are not exported in prl suppressor strains because they fold rapidly and escape recognition by SecB, while secretory proteins, even those lacking a signal sequence, fold more slowly, allowing time for SecB recognition and binding [5, 13]. SecB binds to a variety of unfolded proteins in vitro [24, 25], although binding appears to be more selective in vivo [26]. This selectivity may be based on the slower folding characteristics of secretory proteins.
Therefore, we proposed previously that if a cytoplasmic protein contains a mutation that slows its rate of folding, SecB will recognize and bind to that mutant protein. Binding of SecB would result in targeting of the unfolded protein to the translocation apparatus. According to the proofreading model, in a wild type strain, SecE and SecY would reject the unfolded protein due to lack of a signal sequence. In a Prl suppressor strain, however, the proofreading function would be compromised and the mutant protein would be exported [8]. In the experiments described here, we tested this hypothesis by examining the export of such a cytoplasmic protein with a folding mutation. The results indicate that slow folding is not sufficient for SecB binding and subsequent targeting to translocase and further, that secretory proteins contain targeting information in addition to the signal sequence and the folding kinetics.