Molecular characterization of wild-type and HSAN2B-linked FAM134B

Family with sequence similarity 134, member B (FAM134B), also known as Reticulophagy regulator 1 (RETREG1), is an ER-phagy receptor involved in ER homeostasis. Congenital mutations in the FAM134B gene have been reported to be associated with hereditary sensory and autonomic neuropathy type 2B (HSAN2B); however, the molecular differences between wild-type and HSAN2B-linked FAM134B are not fully understood.

We prepared several human FAM134B constructs, such as the HSAN2B-linked mutant, and compared their features with those of wild-type FAM134B by transfecting these constructs into FAM134B-deficient Neuro2a cells. Although intrinsic FAM134B protein expression in wild-type Neuro2a cells was affected by the supply of amino acids in the culture medium, the expression of each HSAN2B-linked mutant FAM134B protein was hardly affected by serum and amino acid deprivation. On the other hand, the intracellular localization of GFP-tagged HSAN2B-linked mutants, except for P7Gfs133X, overlapped well with ER-localized SP-RFPKDEL and did not differ from that of GFP-tagged wild-type FAM134B. However, analysis of protein‒protein interactions using the NanoBiT reporter assay revealed the difference between wild-type and C-terminal truncated mutant FAM134B. Furthermore, this NanoBiT assay demonstrated that both wild-type and G216R FAM134B interacted with LC3/GABARAPL1 to the same extent, but the FAM134B construct with mutations near the LC3-interacting region (LIR) did not. Similar to the NanoBiT assay, the C-terminal-truncated FAM134B showed lower ER-phagy activities, as assessed by the cotransfection of GFP-tagged reporters.

We showed that wild-type and HSAN2B-linked FAM134B have different molecular characteristics by transfecting cells with various types of constructs. Thus, this study provides new insights into the molecular mechanisms underlying HSAN2B as well as the regulation of ER-phagy.