Abstract
Purpose :
The goal of this study is to elucidate the underlying mechanism(s) of how SLC4A11 traffics to mitochondrial inner membrane in corneal endothelium (CE). Given the preliminary data showing SLC4A11 does not contain classical N-terminal mitochondrial targeting sequences, we hypothesize that mitochondrial SLC4A11 trafficking is through a “carrier import pathway”, which particularly require cytosolic chaperones to guide the precursors to mitochondria through TOM70 (Translocase of the Outer Membrane).
Methods :
The presence of mitochondrial targeting sequences was examined using in silico analyses (MitoProt, TargetP, iPSORT, and Predotar). In order to validate potential cytosolic chaperones, HA-SLC4A11 immunoprecipitation from whole cell lysate of PS120-hSLC4A11-HA were probed with anti-HSC70, and anti-HSP90. For interfering chaperone function, Slc4a11-WT Mouse Corneal Endothelial Cells (MCEC) and PS120-hSLC4A11-HA were treated with HSP90 inhibitors (18 μM geldanamycin and 500 μM novobiocin) for 6 hours and HSP90 siRNA for 72 hours respectively. Then, mitochondria were purified from the cells and resolved with SDS-PAGE gels.
Results :
in silico analyses reveal that SLC4A11 does not contain the canonical mitochondrial targeting sequence in N-terminal protein region similar to well-known mitochondrial proteins such as mitochondrial uncoupling protein 2 (UCP2) and adenine nucleotide translocator (ANT). We also found that co-immunoprecipitation with HA-SLC4A11 as the bait protein revealed that SLC4A11 and HSP90/HSC70 interactions are present in the cytoplasm, further supporting an interaction between SLC4A11 and HSC70/HSP90. In addition, the transfer of SLC4A11 to mitochondria was disrupted by inhibition of HSP90 using 1) inhibitors (18 μM geldanamycin and 500 μM novobiocin) and 2) siRNA knockdown. Interestingly, the distribution pattern of mitochondrial SLC4A11 is most closely linked to the TOM70-dependent preprotein, Adenine nucleotide translocator (ANT), but not to the TOM20-dependent preprotein, Rieske iron-sulfur protein (ISP).
Conclusions :
Our data indicates that HSP90 and/or HSC70 are the potential chaperones that guide SLC4A11 into the mitochondrial inner membrane. Investigating the underlying mechanism of mitochondrial SLC4A11 trafficking is of fundamental importance in understanding its potential (patho-) physiological role in CE.
This is a 2020 ARVO Annual Meeting abstract.