Uncovering the molecular mechanisms that define specificity of homologous machineries using transient interactomics

• Applicant: Emma Fenech, Ph.D.
• Subject Area: Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 551266484

The endoplasmic reticulum (ER) is the largest organelle in the cell and performs a wide array of essential functions. Almost every one of these functions is controlled by homologous protein machineries that reside in this organelle. These pairs, or families, of protein homologs provide each other with back-up functionality. However, beyond redundancy, they have additional roles and can be distinguished from one another by unique functions and regulatory mechanisms. Since it has been challenging to map these specific features, many of the homolog pairs remain incompletely characterised and we lack understanding as to how they work in tandem. One way to gain insight into the mechanisms underlying homologous protein specificity is to understand their unique interactions with other proteins. Protein-protein interactions (PPIs) can either be stable or transient, and together they inform on protein function and regulation. Until recently, the nature of transient interactions (e.g. enzyme-substrate) has made them challenging to capture and identify. However, the recent advent of proximity-labelling (PL), which utilises modified biotin ligases to label proteins within a 10nm radius, has made transient PPI identification much more accessible. During my PhD I became an expert in mapping stable PPIs for large protein families in mammalian cells, and in my postdoctoral research I established advanced methods to systematically map transient PPIs in yeast. To build further on this work and advance our understanding of ER functions, I aim to reveal the mechanistic drivers of selectivity by uncovering transiently-interacting clients, substrates, and regulators of ER-resident homologs on an unprecedented scale. In Aim 1, I will discover unique, condition-specific regulators of membrane contact site tethering homologs by widening the scope of these PL tools. This will be done by creating new yeast strain collections (known as libraries) in which every gene in the genome is tagged with PL enzymes that are tailored to probe contact site biology. In Aim 2, I will uncover the molecular determinants of substrate specificity for quality control machineries on a whole-organelle level by combining different PL libraries with high-throughput mass spectrometry approaches to gain deeper insights. In Aim 3, I will take these tools and systems to the next level in higher eukaryotes, to allow for accurate and efficient identification of unique regulators and substrates for the KDEL retrieval receptor homologs, which have diversified from yeast to human cells. Together, the outlined work will transform our understanding of how redundancy and specialization of ER-resident factors help maintain the plethora of functions of the largest cellular organelle. In addition to providing an important scientific and technical resource, our proteomics studies will serve as a wide basis for collaborations while allowing me to focus on selected key questions in ER biology and human disease.

• DFG Programme: Independent Junior Research Groups
• Major Instrumentation: Benchtop microbial array manipulator
• Instrumentation Group: 1060 Dilutoren, Pipettiergeräte, Probennehmer