Mitochondrial Targeting of the Ammonia-Sensitive Uncoupler SLC4A11 by the Chaperone-Mediated Carrier Pathway in Corneal Endothelium

Abstract

Purpose SLC4A11, an electrogenic H + transporter, is found in the plasma membrane and mitochondria of corneal endothelium. However, the underlying mechanism of SLC4A11 targeting to mitochondria is unknown. Methods The presence of mitochondrial targeting sequences was examined using in silico mitochondrial proteomic analyses. Thiol crosslinked peptide binding to SLC4A11 was screened by untargeted liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. Direct protein interactions between SLC4A11 and chaperones were examined using coimmunoprecipitation analysis and proximity ligation assay. Knockdown or pharmacologic inhibition of chaperones in human corneal endothelial cells (HCECs) or mouse corneal endothelial cells (MCECs), ex vivo kidney, or HA-SLC4A11–transfected fibroblasts was performed to investigate the functional consequences of interfering with mitochondrial SLC4A11 trafficking. Results SLC4A11 does not contain canonical N-terminal mitochondrial targeting sequences. LC-MS/MS analysis showed that HSC70 and/or HSP90 are bound to HA-SLC4A11–transfected PS120 fibroblast whole-cell lysates or isolated mitochondria, suggesting trafficking through the chaperone-mediated carrier pathway. SLC4A11 and either HSP90 or HSC70 complexes are directly bound to the mitochondrial surface receptor, TOM70. Interference with this trafficking leads to dysfunctional mitochondrial glutamine catabolism and increased reactive oxygen species production. In addition, glutamine (Gln) use upregulated SLC4A11, HSP70, and HSP90 expression in whole-cell lysates or purified mitochondria of HCECs and HA-SLC4A11–transfected fibroblasts. Conclusions HSP90 and HSC70 are critical in mediating mitochondrial SLC4A11 translocation in corneal endothelial cells and kidney. Gln promotes SLC4A11 import to the mitochondria, and the continuous oxidative stress derived from Gln catabolism induced HSP70 and HSP90, protecting cells against oxidative stress.

The H+ Transporter SLC4A11: Roles in Metabolism, Oxidative Stress and Mitochondrial Uncoupling

“…Parents of CHED children who are heterozygous for SLC4A11 mutations also appear to be at risk of developing FECD [ 34 ]. No other human disorders have been definitively associated with SLC4A11; however, there are some reports from animal models that suggest kidney involvement (polyuria) [ 32 , 35 , 36 , 37 ] and submandibular gland dysfunction [ 38 ], which is consistent with the strong expression of SLC4A11 in these tissues.…”

“…Inhibiting the HSP90 or HSC70 chaperone pathways in transfected cells and WT MCEC in turn inhibited the delivery of SLC4A11 to mitochondria, consistent with reduced glutamine dependent OCR, reduced ammonia production, and increased mitochondrial superoxide production. Moreover, Choi et al [ 37 ] showed reduced OCR in mitochondria isolated from SLC4A11 KO MCEC and mouse kidney following inhibition of chaperone activity, confirming that the metabolic alterations seen in SLC4A11 deficient cells are not due to non-mitochondrial effects. Heat shock protein expression is typically increased by oxidative stress.…”

“…Proteins are directed to mitochondria either by an N-terminal pre-sequence signal peptide or by a chaperone-mediated pathway involving cryptic internal targeting sequences. Choi et al [ 37 ], using in silico analysis and mass spectrometry of cross-linked proteins from PS120 transfected fibroblasts, determined that SLC4A11 does not have a mitochondrial pre-sequence, but utilizes a chaperone pathway. Both HSP90 and HSC70 participate in guiding SLC4A11 to the mitochondrial outer membrane complex, TOM70, which transports proteins into the intermembrane space, followed by inner membrane chaperones (TIMs) that guide the protein to the inner membrane.…”

“…Heat shock protein expression is typically increased by oxidative stress. Glutamine causes oxidative stress in corneal endothelial cells, so it was not surprising that Choi et al [ 37 ] found that glutamine induced expression of HSP90, HSC70, and SLC4A11 in human corneal endothelial cells, suggesting that SLC4A11 is also an oxidative stress responsive protein.…”

“…Concomitantly, there was a reduced nuclear factor erythroid-related factor-2 (NRF2) expression, the master regulator of these and other antioxidant genes. Guha et al [ 27 ] showed that oxidative stress increased SLC4A11, NRF2, and HO-1 expression in human corneal endothelial cells, consistent with glutamine causing increased SLC4A11 expression [ 37 ]. Knockdown of WT SLC4A11 by siRNA increased ROS formation, decreased mitochondrial activity, and impaired increased expression of NRF2, HO-1, and NQO1 in response to oxidative stress.…”