Periodic Reporting for period 3 - CRYTOCOP (Coat assembly and membrane remodelling: understanding regulation of protein secretion)
Reporting period: 2022-11-01 to 2024-04-30
This project is focused on understanding the mechanisms of protein trafficking between intracellular compartments.
In particular we study the transport of newly synthesised proteins from the Endoplasmic Reticulum to the Golgi apparatus. This is a fundamental step in eukaryotic cell biology, and it is mediated by a protein complex name COPII, which forms a coat around the transport vesicles while concentrating cargo inside them.
A third of all proteins are transported into COPII-coated vesicles, and disruption of this for certain cargo proteins is linked to a number of genetic diseases.
Despite this, there are many unanswered questions with regards to the molecular mechanisms of COPII: how does it assemble to bend membranes? How is the process regulated? What is the morphological variability of COPII-coated vesicles?
The objectives of this proposal are to answer these questions using a multiscale approach, ranging from high resolution details of in vitro reconstituted reactions, to FIB/SEM studies of the COPII coat in cells.
In particular we study the transport of newly synthesised proteins from the Endoplasmic Reticulum to the Golgi apparatus. This is a fundamental step in eukaryotic cell biology, and it is mediated by a protein complex name COPII, which forms a coat around the transport vesicles while concentrating cargo inside them.
A third of all proteins are transported into COPII-coated vesicles, and disruption of this for certain cargo proteins is linked to a number of genetic diseases.
Despite this, there are many unanswered questions with regards to the molecular mechanisms of COPII: how does it assemble to bend membranes? How is the process regulated? What is the morphological variability of COPII-coated vesicles?
The objectives of this proposal are to answer these questions using a multiscale approach, ranging from high resolution details of in vitro reconstituted reactions, to FIB/SEM studies of the COPII coat in cells.
We have used cryo-electron tomography and subtomogram averaging to obtain high-resolution insights into COPII coat assembled on membranes in vitro. This revealed a complex and partially redundant network of interactions participate to coat assembly and drives membrane deformation, leading us to propose a model for regulation of membrane morphology by the coat (Hutchings et al 2021).
We have also made good progress on analysing the structure of the COPII coat with cargo proteins bound, and looking into cells to visualise the secretory apparatus (unpublished).
We have also made good progress on analysing the structure of the COPII coat with cargo proteins bound, and looking into cells to visualise the secretory apparatus (unpublished).
We recently pushed the resolution of the coat by tomography and subtomogram averaging to achieve 3.8A which is beyond what most subtomogram averaging projects have achieved to date. We expect that out newly developed high resolution pipelines will be applicable to our other projects to give us novel insights into coat assembly and regulation in vitro and in cells.