S. K. Flores, Y. Deng, Z-M. Cheng, Y. Qin, P. L.M. Dahia. Germline mutations in the transmembrane protein-encoding gene, TMEM127, have been associated with pheochromocytomas (PHEOs), highly hereditary tumors of the adrenal medulla. The aim of the study was to characterize the functional consequences of several germline TMEM127 mutations detected in patients with PHEOs by investigating the subcellular localization and protein stability of the resulting mutant TMEM127 proteins. GFP-tagged wild-type (WT) and mutant TMEM127 plasmids were generated and transiently transfected into HEK293FT cells. Cells were collected for confocal images and/or Western blot at 24, 48 and 72 hours. TMEM127-TMEM127 interactions were investigated by use of multiple tags and pulldown assays. Missense mutations (N=4) that occurred outside of putative transmembrane domains resulted in mutant proteins that retained a punctate pattern similar to the WT TMEM127 indicative of retention of membrane localization. Missense mutations (N=5) and frameshift mutations (N=2) that occurred within or disrupted putative transmembrane domains resulted in mutant proteins with a diffuse, cytoplasmic pattern suggestive of loss of membrane localization. Moreover, mutant proteins with a diffuse, cytoplasmic pattern were unstable and rapidly degraded within 72 hours compared to WT TMEM127. We identified TMEM127-TMEM127 interactions suggesting that this protein forms dimers or multimers. Membrane localization of TMEM127 is necessary for its stability and, likely, its function. Loss of membrane localization is associated with mutations disrupting putative transmembrane domains. Although mutations occurring outside of putative transmembrane domains retain membrane localization, another function, such as binding to an associated protein, may be affected and requires further investigation. Furthermore, our results support a novel, 4th functional transmembrane domain and TMEM127-TMEM127 interactions, suggesting that dimerization (or multimerization) may be relevant for its function.