Weak pulling forces exerted on Nin-orientated transmembrane segments during co-translational insertion into the inner membrane of Escherichia coli

•We measured co-translational pulling forces on Nin-facing transmembrane segments.•The pulling forces are weaker than for Nout-orientated transmembrane segments.•The biphasic shape of force profiles from Nin- and Nout-segments is preserved.

Transmembrane helices (TMHs) in membrane proteins can be orientated with their N-terminus towards the cytoplasm (Nin), or facing the non-cytoplasmic side (Nout). Most membrane proteins are inserted co-translationally into membranes, aided by Sec-type translocons. Since the final orientation of Nin- and Nout-orientated TMHs differs, they could also interact differently with the translocon and the surrounding membrane during insertion. We measured pulling forces exerted on Nin-orientated TMHs during co-translational insertion into the inner membrane of Escherichia coli. Our results demonstrate that Nin-orientated TMHs experience a weaker pulling force but retain the overall biphasic force profile seen previously for Nout-orientated TMHs (Ismail et al., 2012 [1]).