The ubiquitous Sec machinery facilitates protein movement across or integration of proteins into the cytoplasmic membrane in a post- or cotranslational manner, respectively. In vivo, the bacterial core SecYEG translocon associates with additional membrane components, SecDF-YajC and YidC, forming a complex referred to as the holo-translocon (HTL). A recent breakthrough came from the isolation of a stable complex comprising all seven subunits, enabling analysis of HTL function and interactions between its constituents. HTL's activity in protein secretion and membrane protein insertion was analysed both in the presence and absence of the proton-motive force (PM F). Findings presented here suggest that the HTL supports both processes. Interestingly, it appears less efficient in protein secretion than SecYEG alone, but more responsive to the PMF. Nevertheless, the HTL is more effective at membrane protein incorporation for the majority of substrates analysed in this study. It also appears more efficient at assembly of membrane protein complexes investigated here. These findings suggest that the HTL complex is preferential for membrane protein insertion, whereas SecYEG is more effective in protein secretion. An activated conformation of SecYEG was also investigated in this study. For this purpose, cross-links were designed within the channel based on the structure representing its unlocked state when bound to a signal sequence. Fixing the channel in two different conformations, representing unlocking of the SecY channel and displacement of the plug domain, resulted in its increased activity in protein secretion. However, only the latter conformation had an effect on membrane protein insertion, which suggests major differences in the activation mechanism between these processes. Findings presented here have helped in the understanding of the recent structure of the HTL. This structural information together with functional studies reported here address unknown aspects of the fundamental problem in biology: membrane protein insertion, folding and assembly.