MyD88 polymerization and association to cellular membranes in a yeast heterologous model

MyD88 is a key mediator of Toll-like receptor (TLR) signaling, orchestrating the innate immune response upon stimulation by pathogen-associated molecular patterns (PAMPs). Structurally, MyD88 consists of a Death domain (DD), a 20-amino acid N-terminal extension, and an intermediate (INT) region that connects it to a Toll/Interleukin-1 receptor (TIR) domain. At the core of the signaling complex known as myddosome, MyD88 undergoes homopolymeric interactions to propagate the signal. In this study, we use Saccharomyces cerevisiae as a heterologous model to assess the contribution of individual MyD88 domains to self-interaction and subcellular localization. In yeast, MyD88 localizes to endoplasmic reticulum-mitochondria encounter sites (ERMES). Here, we show that its DD is sufficient for attachment to the ERMES. Deletion of its 20 N-terminal residues increased MyD88 stability, shifting its aggregation pattern from patches to filaments. In contrast, a chimeric MyD88 variant bearing the plasma membrane-binding N-terminal extension of TIRAP, another TLR4-associated myddosome component, exhibited diffuse mitochondrial distribution. Moreover, we found that the ERMES-associated dynamin-like protein Dnm1, involved in mitochondrial fission, played a crucial role in MyD88 expression in yeast. On the other hand, the MyD88 TIR domain alone accumulated at lipid droplets in yeast, and its overexpression led to growth impairment and mitochondrial condensation. These findings suggest that MyD88 association with cellular membranes promotes self-assembly, a process essential for functional TLR signaling. Additionally, we adapted a tripartite GFP system to titrate MyD88 homopolymerization in yeast. Using this system, we observed that the oncogenic L252P mutation significantly reduced MyD88 ability to self-interact.