The ESX-1 secretion system is a type VII secretion system that is necessary for the virulence of M. tuberculosis and select other pathogenic mycobacteria. We sought to determine how protein targeting and protein modification contribute to the function of the ESX-1 secretion system. Accordingly, we identified the amino acids within the targeting sequence of the ESX-1 substrate EspC which are necessary for interaction with the ATPase EccA1 and for secretion of EspC. We also characterized the ESX-1 protein EspN, which we hypothesize plays a role in acetylation of ESX-1 substrates. EspN was shown to interact with the M24 metallopeptidase PepQ from M. tuberculosis. We have created a possible model for the interaction of PepQ and EspN and subsequent acetylation of ESX-1 proteins. Here, we gain a fuller understanding of the role that protein modification and targeting play in the function of the ESX-1 secretion system and subsequent mycobacterial virulence.