SecYEG Proteoliposomes Catalyze the Δϕ-Dependent Membrane Insertion of FtsQ

Abstract

In Escherichia coli, the insertion of most inner membrane proteins is mediated by the Sec translocase. Ribosome-bound nascent chains of Sec-dependent inner membrane proteins are targeted to the SecYEG complex via the signal recognition particle pathway. We now demonstrate that the signal recognition particledependent co-translational membrane targeting and membrane insertion of FtsQ can be reconstituted with proteoliposomes containing purified SecYEG. SecA and a transmembrane electrical potential are essential for the translocation of the large periplasmic domain of FtsQ, whereas co-reconstituted YidC has an inhibitory effect. These data demonstrate that membrane protein insertion can be reconstituted with a minimal set of purified Sec components.

A Derivative of Lipid A Is Involved in Signal Recognition Particle/SecYEG-dependent and -independent Membrane Integrations

“…In this system, we could reproduce three different modes of membrane protein integration as follows: (i) the SRP/SR-and SecYEG-dependent integration of polytopic membrane proteins such as MtlA; (ii) the SRP/SR-and SecYEG-dependent integration of membrane proteins with SecA-requiring periplasmic regions such as Momp2; and (iii) the Sec-and SRP/SR-independent integration of proteins such as the M13 procoat. Although integration by the second and third mode had been described previously using (proteo)liposomes (8,14,51,52), the successful integration of polytopic membrane proteins into proteoliposomes in an SRP/ SR-and SecYEG-dependent manner has not been reported before. In addition to showing the various dependences on known assembly factors, we identified a novel integration stimulating activity.…”

“…Likewise, YidC was rather inhibitory as to the integration into proteoliposomes of FtsQ, a Momp2-type membrane protein (52). Whereas YidC is essential for the integration of Sec-independent membrane proteins (11-13, 15), integration of lactose permease (LacY) can also occur in the absence of YidC resulting, however, in a non-native conformation of LacY (19).…”

Conserved Negative Charges in the Transmembrane Segments of Subunit K of the NADH:Ubiquinone Oxidoreductase Determine Its Dependence on YidC for Membrane Insertion

“…YidC has been shown to contact the TMSs of numerous integral membrane proteins in various cross-linking studies (24,42,43), and yet YidC is dispensable in the insertion of most of these proteins (26,44,45). Using in vitro insertion into proteoliposomes, we show that NuoK minimally requires both YidC and SecYEG for insertion.…”

“…CyoA, however, does not contain any membrane-negative charges. YidC is not essential for the insertion of FtsQ (26) and LepB (49), even though interaction between these proteins and YidC during membrane insertion has been observed (1,24,42,43,50). FtsQ does not contain any membrane-embedded charges, whereas LepB contains a glutamate in TM2.…”

“…This yielded plasmids pETNuoK E36K, pETNuoK E72K, and pETNuoK E36K,E72K, and the mutations were confirmed by sequencing. Plasmid pBSKFtsQ was used for in vitro expression of FtsQ (26). DNA manipulations were performed using E. coli DH5␣ to maintain plasmids and constructs.…”