Mechanisms of co-translational quality control during the synthesis of CFTR

• Applicant: Débora Broch Trentini Schmidt, Ph.D.
• Subject Area: Biochemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 540625656

The synthesis of transmembrane proteins, representing circa 20–30% of the mammalian proteome, entails overcoming significant biophysical challenges. Hydrophobic protein segments must be shielded from the crowded aqueous cytosol as well as transported past the highly polar surface of the membrane into the hydrophobic core of the lipid bilayer. Therefore, the majority of transmembrane proteins are synthesized at the surface of the ER, where they are co-translationally inserted and assembled into the membrane as they emerge from the ribosome. As a result, translation dynamics plays an important role in transmembrane protein biogenesis. Our previous work revealed that large multi-pass membrane proteins are particularly susceptible to co-translational degradation by the Ribosome-associated quality control (RQC) pathway, which is recruited to ribosomes that stalled during translation. To study how translation dynamics influence quality control processes engaged during membrane protein synthesis, we have chosen CFTR as a model substrate. This is a large plasma membrane protein that functions as a chloride anion channel in epithelial cells. CFTR is also the causative agent of Cystic Fibrosis, a prevalent life-threatening genetic disorder. Our preliminary experiments demonstrated that a sizable portion of CFTR translation events are aborted before protein synthesis is completed, with concomitant degradation of the unfinished polypeptide chain by the RQC pathway. We also observed that the incidence of abortive CFTR translation increased upon inactivation of GCN1, a ribosome collision sensor implicated in regulation of translation dynamics, mRNA turnover, and stress signaling. In the present project, we aim to elucidate the co-translational quality control mechanisms engaged upon failed CFTR translation in human cells, with special emphasis on the interplay between RQC and GCN1. Using a combination of fluorescence-based reporters and biochemical analyses, we will characterize the quality control factors recruited to CFTR-translating ribosomes and will delineate their impact on CFTR biogenesis. We anticipate that our research plan will not only reveal novel fundamental co-translational quality control processes, but also point to new venues for therapy of Cystic Fibrosis and of other genetic diseases caused by problems in transmembrane protein synthesis.

• DFG Programme: Research Grants