Flexibility in targeting and insertion during bacterial membrane protein biogenesis Edwin van Bloois, Corinne M. ten Hagen-Jongman, Joen Luirink * Department of Molecular Microbiology, Institute of Molecular Cell Biology, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands Received 30 July 2007 Available online 20 August 2007 Abstract The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Membrane protein assembly; Protein targeting; Ribosome; SRP; YidC Sec-translocon; Escherichia coli The biogenesis of Escherichia coli IMPs is accomplished in three consecutive steps: (i) membrane targeting, (ii) insertion into the lipid bilayer, and (iii) folding and assem- bly into the final functional structure. The biogenesis of only a few IMPs has been studied in detail pointing at mul- tiple versatile pathways involving assistance by different proteinaceous factors [1,2]. Targeting of most IMPs ana- lyzed thus far requires a conserved and essential system comprising the signal recognition particle (SRP) and its receptor FtsY [3]. The E. coli SRP consists of the signal binding protein Ffh and the 4.5S RNA. The SRP binds to hydrophobic targeting signals present in nascent IMPs. The ribosome nascent chain–SRP complex is then trans- ferred to the Sec-translocon via FtsY. The Sec-translocon is a conserved heterotrimeric com- plex, consisting of the IMPs SecY, SecE, and SecG and functions as a protein conducting channel for both secre- tory proteins and IMPs [1]. The ATPase SecA is peripher- ally associated with the SecYEG complex and drives the translocation of secretory proteins and larger periplasmic domains of IMPs. Additionally, the proton motive force (pmf) is required for protein translocation and many IMPs require the pmf for insertion and assembly [2]. YidC was recently identified as an essential IMP that is in part associated with the Sec-translocon [1]. It plays a pivotal but poorly defined role downstream of the Sec-tran- slocon in later steps of IMP biogenesis such as the recogni- tion and lateral transfer of TMs from the Sec-translocon (FtsQ, Lep), the assembly of TMs (MtlA) and folding of the IMPs into their native structure (LacY). YidC also operates as a separate Sec-independent insertase. This alternative integration pathway is used by a few relatively simple IMPs, including the small phage coat proteins M13 and Pf3, and the endogenous IMPs F o c and MscL. The different targeting and integrating factors such as SRP, Sec(A)YEG, and YidC can be envisioned as modules. Different combinations of these modules result in the differ- ent membrane biogenesis pathways for specific IMPs [2]. The use of the different targeting and integration modules is probably primarily determined by specific structural fea- tures of an IMP. Only a limited collection of IMPs has been analyzed in detail with respect to the requirements 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.08.053 * Corresponding author. Fax: +31 20 5987155. E-mail address: joen.Luirink@falw.vu.nl (J. Luirink). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 362 (2007) 727–733