Sequences involved in the different cellular localizations of mitochondrial and nuclear TR alpha 1 proteins
Résumé
The TR alpha 1 transcript encodes four proteins by the use of internal AUG. Whereas proteins with molecular weights of 47 and 30 kDa are localized in the nucleus, proteins with molecular weights of 43 (p43) and 28 kDa (p28) display a mitochondrial localization and act as T3 mitochondrial receptors. However, the differential location of integral and truncated TR alpha 1 proteins remains poorly understood. We performed in organello import studies with full-length or truncated TR alpha 1 proteins. P43 import occurred according to an atypical import process: absence of peptide signal cleavage, insensitivity to mitochondrial membrane potential or ATP stores. As expected, in contrast to p43 and p28, p47 (nuclear receptor) and p30 were not imported into the organelle. By using deletion mutants of TR alpha 1 proteins, we found that two sequences located in the ligand binding domain are involved in the mitochondrial targeting of TR proteins: helix 5 and helices 10-11. Fusion of these sequences to EGFP induces the mitochondrial import of this cytosolic protein; conversely deletion of helices 5 and 10-11 abrogates translocation of p43 and p28 into mitochondria. As all TR alpha 1 proteins share these two sequences, we studied the importance of the N-terminal part of each protein. We found that the occurrence of negative charges in the first 8 amino acids abrogates the mitochondrial import. In agreement with this possibility, deletion of 12 N-terminal amino acids of p43 revealing a stretch of negatively charged amino acids blocks the p43 mitochondrial import. In conclusion, all TR alpha 1 proteins display mitochondrial import sequences. However, the Nterminal part of each protein allows or abrogates functionnality of the two import sequences and plays a major role for the specific cell localization of TR alpha 1 proteins These data explain the occurrence of mitochondrial and nuclear receptors encoded by a common transcript at the origin of different T3 action pathways.