Deciphering post-translational insertion of tail-anchored protein into the ER membrane – MembraIN

Tail-anchored (TA) proteins, which have a single hydrophobic transmembrane domain (TMD) near their C-terminus, play important roles in a wide range of molecular and cellular functions in every organism. Special machinery is required to shield the TMD against hydrophilic cytosol after protein synthesis and insert it in the endoplasmic reticulum (ER) membrane. This shielding/insertion process has recently sparked attention in cell biology and several pathways, called post-translational pathways, have been discovered. However, the actual molecular mechanisms remain largely unknown. The most significant and fundamental unanswered question is how a shaft is created on the membrane to bypass the energy barrier coming from the crossing of the hydrophilic lipid head group of the membrane by the highly hydrophobic TMD.
I believe, as acknowledged by many authors in the field, that this question can only be addressed by an interdisciplinary approach between soft-matter physics and cell biology. This is exactly what I plan for this project. I will use my high-end microfluidic setup to fully describe, at the molecular level, how TA proteins are inserted into the ER membrane and how the machinery is recycled. This 3D printing-based setup produces the most accurate mimic of physiological membranes, which enables the full recapitulation and characterization of any in vivo membrane process through simultaneous optical and electrical measurements. My preliminary experiments on the most common insertion machinery successfully validate the use of this assay to decipher post-translational pathways. In this project, I will recapitulate two complete insertion and recycling processes, called the GET and the EMC pathways, and dissect their molecular mechanism, which will notably address how the energy barrier is bypassed. The integrity of those shielding/insertion recapitulations will be demonstrated by the artificial reproduction of TA protein insertion using synthetic DNA-TMD hybrids.