Title:
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Activity of the holo-translocon and its individual components in protein secretion and membrane protein insertion
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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.
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