Increased Polyubiquitination and Proteasomal Degradation of a Munc18-1 Disease-Linked Mutant Causes Temperature-Sensitive Defect in Exocytosis

Sally Martin, Andreas Papadopulos, Vanesa M. Tomatis, Emma Sierecki, Nancy T. Malintan, Rachel S. Gormal, Nichole Giles, Wayne A. Johnston, Kirill Alexandrov, Yann Gambin, Brett M. Collins, Frederic A. Meunier

Munc18-1 is a critical component of the core machinery controlling neuroexocytosis. Recently, mutations in Munc18-1 leading to the development of early infantile epileptic encephalopathy have been discovered. However, which degradative pathway controls Munc18-1 levels and how it impacts on neuroexocytosis in this pathology is unknown. Using neurosecretory cells deficient in Munc18, we show that a disease-linked mutation, C180Y, renders the protein unstable at 37°C. Although the mutated protein retains its function as t-SNARE chaperone, neuroexocytosis is impaired, a defect that can be rescued at a lower permissive temperature. We reveal that Munc18-1 undergoes K48-linked polyubiquitination, which is highly increased by the mutation, leading to proteasomal, but not lysosomal, degradation. Our data demonstrate that functional Munc18-1 levels are controlled through polyubiquitination and proteasomal degradation. The C180Y disease-causing mutation greatly potentiates this degradative pathway, rendering Munc18-1 unable to facilitate neuroexocytosis, a phenotype that is reversed at a permissive temperature.