Purification of the Translocon-associated protein complex in a near-native state.

Abstract

The ER translocon machinery is a macromolecular assembly, responsible for the admission of membrane proteins into the secretory pathway. During co-translational translocation, the translocon-associated protein complex (TRAP) interacts as a constituent co-factor, with Sec61 -the main component of the ER translocon machinery. The detailed structural determination of the translocon core both in a native and an isolated state has provided us significant insight regarding the structure of TRAP in this context; even so, little is known about its function(s). Hence, it is important to obtain an atomic model of TRAP to further understand its intracellular contribution. In this study, we managed to establish a protocol for large-scale production of the complex in an isolated state in order for its structural determination via cryoEM SPA to be feasible. A native-nanodisc-forming copolymer and different detergents were tested for the encapsulation of a stable TRAP complex from the ER membrane. The purification of the complex via GDN-PMAL/C8 seemed to not disrupt its integrity and thus led to an initial 7.53 Å 3D model of isolated TRAP. Even thought we could not conclude about a potential differentiation in the complex’s configuration outside the ER translocon machinery context based on these data, our optimized sample preparation could be exploited by future cryoEM studies to solve the structure of TRAP in high resolution and thus comprehend its biological relevance.