Decoding the molecular mechanisms of membrane protein targeting and insertion

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The spatial and temporal coordination of protein trafficking is essential in all cells and in particular important for aggregation-prone membrane proteins. The enormous functional heterogeneity of membrane proteins is reflected by their structural diversity, which directly impacts on their insertion mechanism into the membrane. In bacteria, the majority of membrane proteins are co-translationally recognized by the signal recognition particle (SRP), which delivers ribosome-associated nascent proteins (RNCs) to either the SecYEG translocon or the YidC insertase for insertion. How SRP discriminates between both insertion sites is unknown. The SRP receptor FtsY coordinates the transfer of RNCs from SRP to SecYEG, but recent data indicate that FtsY is not required for YidC targeting. This would reflect an unprecedented non-canonical targeting reaction by SRP. Such a role of SRP is also expected during targeting of special membrane proteins, like tail-anchored proteins. Exploring these atypical reactions of SRP further and revealing the interplay of SRP with general chaperone systems will further expand our perception of the immense plasticity of protein targeting systems. The SecYEG translocon forms a highly dynamic protein complex, but the exact function of many of the already known partner proteins and those that we have newly identified is unknown. Our recent data show that some of the accessory proteins reach deeply into the periplasmic vestibule of SecY, which could indicate that they can execute a pulling force on substrates. Different to the dynamic SecYEG translocon, YidC is considered to function as solitary unit during membrane protein insertion. However, a detailed exploration of potential YidC partner proteins in vivo and in vitro is missing. Identifying partner proteins and investigating their function will be in particular revealing in light of recent reports that identified YidC homologues in the archaeal membrane and the ER membrane. This should allow to define similarities and differences of the YidC network in different cellular membranes. Translation-independent targeting of membrane proteins adds yet another variation to bacterial protein insertion mechanisms that needs to be addressed. By combining single molecule in vivo approaches for monitoring mRNA localization in living cells with biochemical tools for determining interaction partners of mRNAs, we will provide insight into this ill-defined process. In summary, this proposal aims to explore the diversity of membrane protein targeting systems, reveal their interaction with general chaperone systems and explore the dynamic assemblies of the SecYEG and YidC insertion sites. This will provide the conceptual framework for understanding the plasticity of protein transport systems that sustain cell viability in pro- and eukaryotes.