Eukaryotic cells have lipid-enclosed microenvironments, ‘organelles’, that provide suitable environments for specific biochemical reactions. The Endoplasmic Reticulum (ER) is the site, where a major portion of protein synthesis occurs. Approximately one third of the human genome is part of the secretory pathway: they are synthesized at the ER membrane, mature further in the ER and are transported to their eventual destination, such as the exterior of the cell, the plasma membrane or intracellular organelles. The ER is equipped with an intricate protein network to govern the biogenesis and homeostasis of this large and diverse group of ‘secretory pathway’ proteins. The molecular mechanisms of how this machinery facilitates protein biogenesis and degradation are largely unknown to date. In this project, we use an integrative approach with cryo-electron tomography in its core to study the BiogENesis and Degradation of ER proteins (BENDER).
The overall goal of the BENDER project is to study the molecular architecture of the ER biogenesis and degradation machinery ‘in action’: 1) We focus on the static structure of the entry to the ER and secretory pathway, the ER translocon complex. This ‘swiss army knife’ can team up with accessory modules that meet the requirements of its various substrates and the state of the cell, 2) We study the maturation of proteins within the ER and 3) focus on how the ER maintains protein homeostasis under conditions of stress. To achieve these goals, we develop advanced computational methods for cryo-electron tomography that allow to most effectively distinguish different types of molecules involved in protein biogenesis and degradation as well as their different conformational states.
At the conclusion of this project, we have identified multiple intermediate states in the ER translation/translocation pathway, involving several new stoichiometric and substoichiometric components of the translocon. We have solved the structure of the human signal peptidase complex and gained significant new insight the mode of action of this complex that is essential for the maturation of proteins in the ER. We have characterized macromolecular changes that occur in the ER in response to protein-folding stress. In the process we have developed methods to localize specific macromolecules in cellular volumes and statistical analysis of their interactions, revealing protein biogenesis in the larger physiological context.
