Emmanuel Akoto

emmanuel.akoto@bsu.edu

cell-biology , microbiology-molecular-biology , chemistry

Exploring the role of the Proteasome and Vacuole in Ste24-Dependent Translocon Quality Control Maintaining cellular homeostasis relies on regulated proteolysis at the endoplasmic reticulum (ER). Aggregated or prematurely folded proteins can lead to translocon clogging, which is a critical stressor for the ER and has been associated with pathologies such as Type II Diabetes. Understanding the mechanism by which translocon-clogging proteins are degraded is vital for elucidating the pathogenesis of and developing therapies for this disease. The degradation of these proteins is orchestrated through conserved Translocon Quality Control (TQC) mechanisms, with the protease Ste24 playing a pivotal role. The specific mechanisms by which Ste24 substrates are degraded, however, remain elusive. We aimed to discern whether Ste24 facilitates proteasomal or vacuolar degradation for translocon-clogging proteins, utilizing Saccharomyces cerevisiae as a model system and an engineered translocon-clogging protein called “Clogger”. To determine the degradation mechanism by which Clogger is degraded, we inhibited the proteasome and vacuolar degradative pathways with specific pharmacological agents (MG132 for the proteasome and PMSF for the vacuole). Inhibition of the relevant pathway was expected to stabilize the Clogger substrate. To this end, we generated a panel of yeast strains to increase sensitivity to these inhibitors. Our findings demonstrate that the degradation of Clogger mediated by Ste24 occurs independently of both proteasomal and vacuolar pathways, challenging previous models. Notably, fragments of Clogger at the N- and C-termini did not accumulate following inhibition, suggesting that Ste24 may not directly cleave Clogger or cleaves within the epitope tags, preventing detection. These observations imply that Ste24 facilitates the degradation of substrates that aberrantly clog translocons through a distinct mechanism. This study provides critical insights into the functional role of Ste24 in the degradation of translocon-clogging proteins and opens new avenues for investigation of novel mechanisms of translocon unclogging.