Mutant dominant-negative causes protein aggregates degraded via ERAD and prevents normal rhodopsin from proper membrane trafficking.

Dominant mutations in the rhodopsin gene () contribute to 25% of autosomal dominant retinitis pigmentosa (adRP), characterized by photoreceptor loss and progressive blindness. One such mutation, carries a 3-bp deletion, resulting in the loss of one of two isoleucines at codons 255 and 256. Our investigation, using recombinant expression in HEK293 and COS-7 cells, revealed that , akin to the known adRP mutation , induces the formation of rhodopsin protein (RHO) aggregates at the perinuclear region. Co-expression of or with wild-type , mimicking the heterozygous genotype of adRP patients, demonstrated the dominant-negative effect, as all isoforms were retained in perinuclear aggregates, impeding membrane trafficking. In retinal explants from WT mice, mislocalization of labeled adRP isoforms at the outer nuclear layer was observed. Further analysis revealed that RHO aggregates are retained at the endoplasmic reticulum (ER), undergo ER-associated degradation (ERAD), and colocalize with the AAA-ATPase escort chaperone valosin-containing protein (VCP). These aggregates are polyubiquitinated and partially colocalized with the 20S proteasome subunit beta-5 (PSMB5). Pharmacological inhibition of proteasome- or VCP activity increased RHO aggregate size. In summary, RHO exhibits dominant pathogenicity by sequestering normal RHO in ER aggregates, preventing its membrane trafficking and following the ERAD degradation.