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Moonjung Choi, Diego Gabriel Ogando, Shimin Li, Rajalekshmy Shyam, Joseph A Bonanno; SLC4A11 is a mitochondrial protein in corneal endothelium.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2159. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal endothelial dystrophy associated gene SLC4A11 is involved in mitochondrial function. The human SLC4A11 gene encodes three N-terminal transcript variants: SLC4A11-A, -B and -C with a purported to have an intracellular location and the others plasma membrane.Given the effects of SLC4A11 on mitochondrial function, we hypothesize that SLC4A11-A is associated with mitochondria of corneal endothelial cells.
Co-localization of SLC4A11 and mitochondria was examined either by Immunofluorescence with whole cells and by immunoblotting with purified mitochondria from Human Corneal Endothelial Cells (HCEC) and Slc4a11-WT& Slc4a11-KO Mouse Corneal Endothelial Cells (MCEC). In addition, in order to test which one of three SLC4A11 gene transcripts is targeted to mitochondria, we established four stably transfected PS120 cell lines using each plasmid N-terminally tagged with Hemagglutinin(HA) (PS120-hSLC4A11-A,-B, and -C-HA). Sub-mitochondrial localization of SLC4A11 was investigated by graded digitonin treatment of isolated mitochondria and comparison with outer and inner membrane markers.
There was colocalization of endogenous SLC4A11 and MitoTracker CMXRos indicating mitochondrial localization of SLC4A11 in corneal endothelium. We also found that SLC4A11 is present in crude mitochondrial preparations from HCEC and Slc4a11 WT MCEC. Interestingly, isolated mitochondria of all three stably transfected PS120 cell lines appeared to be associated with mitochondria. For digitonin digestion, we found that TOM20 and VDAC, outer mitochondria membrane (OMM) proteins, were lost from the mitochondria at digitonin concentrations between 0.2 and 0.4% (w/v). TIM23 and UCP2, inner mitochondria membrane (IMM) proteins, were released at higher concentrations (0.4 % and more). However, GLS1, GLS2, and HSP60, inner mitochondrial matrix proteins, were slightly released at 0.25% but not fully lost. Interestingly, the distribution pattern of SLC4A11 is most closely linked to the IMM proteins. TEM immunochemistry confirmed an IMM location of SLC4A11.
We found direct expression of SLC4A11 in isolated mitochondrial fractions from corneal endothelial cells, which is unexpected in the light of all previous demonstration of SLC4A11 in corneal endothelium. This localization of SLC4A11 into the mitochondria may support the notion that SLC4A11 is protecting corneal endothelium from mitochondria dysfunction.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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