Abstract
Purpose :
SLC4A11 is an ammonia-sensitive mitochondrial uncoupler suppressing glutamine induced oxidative stress. Our preliminary data indicate that SLC4A11 might traffic to the mitochondria of corneal endothelium through a “chaperone-mediated carrier-pathway”. The purpose of this study is to investigate direct physical interaction between molecular chaperones and SLC4A11.
Methods :
In situ protein interactions between SLC4A11 and either HSP90 or HSC70 was examined and visualized using Proximity Ligation Assay (PLA), which uses a pair of oligonucleotide-labeled secondary antibodies (PLA Probes). Anti-HA was paired with either anti-HSP90 or anti-HSC70 primary antibodies in PS120 fibroblasts transfected with either HA epitope-tagged human SLC4A11 (PS120-hSLC4A11-HA) or empty vector (PS120-EV) cells. When the primary antibodies are in close proximity to each other (< 40nm), PLA-fluorescence spots are quantified using a Zeiss LSM 800 confocal microscope with Airyscan (63X objective, 1.8X zoom). In order to validate whether the chaperones guide SLC4A11 to the mitochondrial surface receptor to import the pore of the translocase of the outer membrane (TOM) complex, mitochondria of the cells were labeled with MitoTracker CMXRos prior to PLAs: 1) HA/TOM70 (outer mitochondrial membrane marker), 2) HA/HSP90, and 3) HA/HSC70. Incubation with no primary antibody and each separate primary antibody was done as negative controls for the PLAs.
Results :
Significant PLA signals of SLC4A11 with HSP90 or HSC70 were observed in PS120-hSLC4A11-HA cells, but not in PS120-EV cells. Furthermore, PLA: HA/TOM70 revealed that SLC4A11 directly binds to the mitochondrial surface receptor, TOM70. The PLA: HA/TOM70 signals were colocalized with the mitochondria labeled with MitoTracker CMXRos. Like the distribution of HA/TOM70 PLA signals to the mitochondria, fewer than half of HA/HSP90 and HA/HSC70 PLA signals closely overlapped mitochondria. More than half of the PLAs signals appeared in the cytoplasm, which indicates where SLC4A11-chaperone complexes are initially recruited.
Conclusions :
We found that HSP90 and/or HSC70 are the chaperones as physical binding partners with SLC4A11, which, in turn, allows SLC4A11 to traffic across the mitochondria.
This is a 2021 ARVO Annual Meeting abstract.