Nascent polypeptides selected for export are synthesized in the cytoplasm by ribosomes and inserted into or translocated across membranes to reach their correct location. Exported proteins delay their folding and remain soluble during their cytoplasmic transit to the membrane. In bacteria, most secretory proteins require additional support from cytosolic chaperones such as trigger factor (TF) and SecB to promote their translocation competence. Here, we investigate the effect of TF and SecB on the folding dynamics and in vitro translocation of secretory and cytoplasmic model proteins PpiA and PpiB, respectively. Global hydrogen—deuterium exchange mass spectrometry (HDX-MS) experiments reveal that SecB delays the folding of slow-folding PpiA proteins but has no effect on fast folders like PpiB. In vitro protein translocation results show that TF inhibits the Sec-dependent translocation of mature PpiA/B and derivative proteins, as well as some secretory preproteins carrying a signal peptide (SP), whereas SecB has no clear effect under the same conditions. However, SecB proves to be dominant over TF in protein translocation in vitro. Finally, for the secretory preprotein proPpiA, SecB prevents SP-induced aggregation. Our findings indicate that the combined properties of signal peptides and mature domains dictate chaperone specificity and translocation efficiency, with both TF and SecB acting in a substrate-specific manner.
A Thu, study studied this question.