Pathogenic

Charcot-Marie-Tooth Disease Type 2P (CMT2P) is an inherited axonal neuropathy caused by dominant mutations in the RING domain of LRSAM1. The precise cellular consequences of these mutations remain unclear. Here, we investigated the subcellular localization of wild-type and RING mutant LRSAM1 in multiple cell models, including patient-derived fibroblasts and neuronal cell lines. Using immunocytochemistry, membrane and lipid droplet fractionation, and colocalization with organelle markers, we found that RING mutant LRSAM1 consistently mislocalized, deviating from its typical diffuse cytosolic pattern to form large perinuclear aggregates. This mislocalization also caused abnormal clustering of the LRSAM1 target protein TSG101 that coaggregated with mutant LRSAM1. Wild-type LRSAM1 predominantly localized to the cytosolic fraction, whereas the mutant protein shifted toward the membranous fraction. Wild-type LRSAM1 was also observed in the lipid droplet fraction, whereas mutant LRSAM1 was absent. Our findings demonstrate that pathogenic RING mutations in LRSAM1 disrupt its subcellular localization and that of its targets. These results highlight protein mislocalization as a pathogenic mechanism in CMT2P, providing new insight into disease etiology.