Decoding the molecular mechanisms of membrane protein targeting and insertion

蛋白质运输的空间和时间协调在所有细胞中是必不可少的，并且对于易于聚集的膜蛋白特别重要。膜蛋白结构的多样性反映了其巨大的功能异质性，结构的多样性直接影响着膜蛋白的插入机制。在细菌中，大多数膜蛋白被信号识别颗粒（SRP）共识别，其将核糖体相关新生蛋白（RNC）递送至SecYEG易位子或YidC插入酶以进行插入。SRP如何区分两个插入位点尚不清楚。SRP受体FtsY协调RNC从SRP转移到SecYEG，但最近的数据表明，FtsY不是YidC靶向所必需的。 这反映了SRP前所未有的非典型靶向反应。在靶向特殊膜蛋白（如尾锚定蛋白）的过程中，也预期SRP的这种作用。进一步探索SRP的这些非典型反应，并揭示SRP与一般分子伴侣系统的相互作用，将进一步扩大我们对蛋白质靶向系统巨大可塑性的认识。SecYEG易位子形成高度动态的蛋白质复合物，但许多已知的伴侣蛋白和我们新发现的蛋白质的确切功能尚不清楚。我们最近的数据表明，一些辅助蛋白深入SecY的周质前庭，这可能表明它们可以对底物执行拉力。与动态SecYEG易位子不同，YidC被认为在膜蛋白插入过程中起孤立单位的作用。然而，缺乏对体内和体外潜在YidC伴侣蛋白的详细探索。鉴定伴侣蛋白并研究它们的功能将特别揭示最近的报道，该报道鉴定了古细菌膜和ER膜中的YidC同源物。这应该允许定义不同细胞膜中YidC网络的相似性和差异性。翻译无关的膜蛋白靶向增加了另一种需要解决的细菌蛋白插入机制的变化。通过结合单分子在活细胞中的方法来监测mRNA的定位与生化工具，以确定相互作用的mRNA的合作伙伴，我们将提供深入了解这个不明确的过程。 总之，本提案旨在探索膜蛋白靶向系统的多样性，揭示它们与一般伴侣系统的相互作用，并探索SecYEG和YidC插入位点的动态组装。这将为理解蛋白质转运系统的可塑性提供概念框架，蛋白质转运系统维持原核生物和真核生物的细胞活力。

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.