Role of the Propilin Leader Peptide in the Maturation

and( 14 C)prolinelabelingconfirmeditsidentitysincetheonlyprolineresiduesofpropilinarefoundwithinthe leader peptide. The in vitro and in vivo propilin-processing reactions proceed similarly in a single polypeptide cleavage step. Furthermore, TraQ dependence is a property of F-pilin maturation specifically rather than a property of the leader peptide. A propilin derivative with an amino-terminal signal sequence generated by deleting codons 2 to 28 required TraQ for processing in vivo. On the other hand, a chimeric protein with the propilin wild-type leader peptide fused to the mature portion of b-lactamase was processed in a TraQ- independentmanner.Thus,despiteitsunusuallength,thepropilinleaderpeptideseemstoperformafunction similar to that of the typical amino-terminal signal sequence. This work suggests that TraQ is not necessary for the proteolysis of propilin and therefore is likely to act as a chaperone-like protein that promotes the translocation of propilin. Bacterial conjugation, mediated by the F plasmid, requires the elaboration of surface pili. These F pili initiate the contact between donor and recipient cells that ultimately leads to F DNA transfer. In the absence of pili, cells are transfer defi- cient. The F pilus is assembled from F-pilin subunits that are present as pools in the cytoplasmic membrane (21, 22). The subunits are derived from the traA gene product (4), a 121- amino-acid (aa) polypeptide that is processed to a 7-kDa pep- tide (51 aa) in the presence of the F-encoded TraQ protein (11, 15, 29, 30). This pilin peptide is further modified by acet- ylation by TraX to yield the mature F-pilin subunit (14, 20). In the preceding paper (12), propilin processing was shown to be dependent on leader peptidase (LepB) as well as on an estab- lished proton motive force. Some data in the preceding paper also indicated that processing occurred as a single polypeptide cleavage event, suggesting no role for TraQ-dependent pro- teolytic activity on the propilin leader peptide. Although the Ala-52 residue was identified by peptide se- quencing to be thefirst residue of F pilin (4), and although we had shown in the study described in the preceding paper that LepB is involved in the cleavage of the propilin peptide (12), little was known about the fate of this peptide. In particular, leaderpeptideprocessingcouldoccurintwosteps:TraQcould initiate a first cleavage within the leader peptide, and LepB would then cleave the exposed signal sequence. To examine this possibility, we developed an in vitro assay and refined our in vivo assay to better detect the leader peptide. We also characterized the TraQ dependence of altered propilins that lacked portions of their leader peptide and constructed various protein fusions to determine the function of the leader peptide.