Updated publication reference for PubMed record(s): 27795391. Pathogenic mycobacteria contain up to five type VII secretion (T7S) systems, ESX-1 to ESX-5, which have central roles in virulence and viability. One of the conserved components of these systems, the serine protease mycosin (MycP), has been shown to be essential for secretion by ESX-1 and ESX-5. However, here we show that protease activity of MycP1 and MycP5 of M. marinum is not essential for ESX-1 and ESX-5 dependent secretion, respectively. This indicates that MycP has a second, so far unknown, function that is essential for T7S. To investigate whether this second role is related to proper functioning of the T7S membrane core complex, we first verified that the ESX-1 membrane complex is of similar composition and size as the previously analyzed ESX-5 complex and that MycP1 and MycP5 are not an integral part of the respective core complex. In contrast, we could not detect ESX-1 and ESX-5 complexes under mycP1 and mycP5 knockout conditions, respectively. Interestingly, the ESX-5 complex could be detected at wild-type levels in the absence of MycP5, when membranes were subjected to crosslinking before detergent solubilization, suggesting that MycP is involved in complex stabilization. Based on our findings we hypothesize that mycosins loosely associate with their respective membrane complex, which is crucial for the full integrity and functioning of the secretion machinery.