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nesis. Here, we show that ectodermal-neural cortex 1 (ENC1), a novel binding partner of sequestosome 1 (p62), negatively regulates autophagy under endoplasmic reticulum (ER) stress. We found that ER stress significantly increases the expression of ENC1 via inositol-requiring enzyme 1 (IRE1)-TNF receptor-associated factor 2 (TRAF2)-c-Jun N-terminal kinase (JNK) pathway. Ectopic expression of ENC1 alone induces the accumulation of detergent-resistant mtHTT aggregates and downregulation of ENC1 alleviates ER stress-induced mtHTT aggregation. Simultaneously, ER stress-induced impairment of autophagy flux is ameliorated by downregulation of ENC1. From immunoprecipitation and immunocytochemical assays, we found that ENC1 binds to p62 through its BTB and C-terminal Kelch (BACK) domain and this interaction is enhanced under ER stress. In particular, ENC1 preferentially interacts with the phosphorylated p62 at Ser403 during ER stress. Interestingly, ENC1 colocalizes with mtHTT aggregates and its C-terminal Kelch domain is required for interfering with the access of p62 to ubiquitinated mtHTT aggregates, thus inhibiting cargo recognition of p62. Accordingly, knockdown of ENC1 expression enhances colocalization of p62 with mtHTT aggregates. Consequently, ENC1 knockdown relieves death of neuronal cells expressing mtHTT under ER stress. These results suggest that ENC1 interacts with the phosphorylated p62 to impair autophagic degradation of mtHTT aggregates and affects cargo recognition failure under ER stress, leading to the accumulation and neurotoxicity of mtHTT aggregates.