The N-terminus of human monocarboxylate transporter 8 is a target of ubiquitin depending proteasomal degradation

Introduction: The monocarboxylate transporter 8 (MCT8) is so far the most specific thyroid hormone (TH) transporter. In humans, MCT8 protein exists either with a short N-terminus of 92 amino acids or with an elongated N-terminus of 166 amino acids. The relevance of these additional residues is still unknown. We hypothesized that the elongated N-terminus could be relevant for transporter expression and/or posttranslational modification. Materials & Methods: To address these questions we generated MCT8 constructs with different length of the N-terminus. Constructs were C-terminally fused to green fluorescent protein and transfected into a polarized (MDCK-1) and a non-polarized (HEK293) cell line to compare potential cell polarity effects in MCT8 expression and localization. Results: All MCT8 constructs showed plasma membrane expression. Thus, the elongated N-terminus of MCT8 appears not to be necessary for correct membrane targeting. However, the MCT8 construct with the elongated N-terminus which contains both translational start sites, methionine 1 and 75, showed the lowest protein expression level and appeared as a double band in Western blot analysis. This double band suggests the presence of both MCT8 constructs containing 92 or 166 N-terminal amino acids. Mutation of methionine 75 to glycine resulted in abrogation of the double band. Furthermore the proteasome inhibitor lactacystin increased the expression of MCT8 with the elongated N-terminus as compared to water control treatment. In addition, immuno-precipitation of MCT8 with the elongated N-terminus revealed elevated ubiquitin binding as compared to the short MCT8 construct. Finally, the mutation of one of the two lysine's (lysine 56 to glycine) within the N-terminus which could be responsible for the enhanced ubiquitin binding of MCT8 with the elongated N-terminus, resulted in a weak binding of ubiquitin and strong expression of the construct. Conclusion: Our data suggest a potential role of the elongated N-terminus of human MCT8 as a target of ubiquitin depending proteasomal degradation.