Functional characterization of germline variants in theTMEM127tumor suppressor reveals novel insights into its membrane topology and trafficking

Abstract

Purpose: To better understand the function of the transmembrane protein TMEM127, a poorly known tumor suppressor gene associated with pheochromocytomas, paragangliomas and renal carcinomas, we evaluated patient-derived germline variants.Methods: Subcellular localization and steady-state levels of 21 tumor-associated, transiently expressed TMEM127 variants were compared to the wild-type protein using immunofluorescence and immunoblot analysis, respectively, in cells genetically modified to lack endogenous TMEM127. Membrane topology and endocytic mechanisms were also assessed. Results:We identified three subgroups of mutations and determined that 15 of the 21 variants (71%), including 9 of 15 missense variants (60%), are pathogenic or likely pathogenic, through loss of membrane binding ability, stability and/or internalization capability. Investigation into an N-terminal cluster of missense variants uncovered a previously unrecognized transmembrane domain, indicating that TMEM127 is a four-, not a three-, transmembrane domain-containing protein. Additionally, a C-terminal variant with predominant plasma membrane localization revealed an atypical, extended acidic, dileucine-based motif required for TMEM127 internalization through clathrinmediated endocytosis. Conclusion:We characterized the functional deficits of several germline TMEM127 variants and identified novel structure-function features of TMEM127, namely, a fourth transmembrane domain and an endocytic motif. These findings will assist in TMEM127 variant interpretation and will help guide future studies investigating the cellular role of TMEM127.1