Vision Science Program, Indiana University, School of Optometry, Bloomington, Indiana, United States.
Invest Ophthalmol Vis Sci. 2021 Sep 2;62(12):4. doi: 10.1167/iovs.62.12.4.
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.
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.
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.
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.
SLC4A11 是一种电致质子转运体，存在于角膜内皮细胞的质膜和线粒体中。然而，SLC4A11 靶向线粒体的潜在机制尚不清楚。
使用计算机分析线粒体蛋白质组学对线粒体靶向序列进行了检测。通过非靶向液相色谱/串联质谱（LC-MS/MS）分析筛选与 SLC4A11 结合的硫醇交联肽。通过免疫共沉淀分析和邻近连接测定法检查 SLC4A11 与伴侣蛋白之间的直接蛋白相互作用。在人角膜内皮细胞（HCECs）或小鼠角膜内皮细胞（MCECs）、离体肾脏或 HA-SLC4A11 转染的成纤维细胞中敲低或药理学抑制伴侣蛋白，以研究干扰线粒体 SLC4A11 转运的功能后果。
SLC4A11 不含有典型的 N 端线粒体靶向序列。LC-MS/MS 分析表明，HSC70 和/或 HSP90 与 HA-SLC4A11 转染的 PS120 成纤维细胞全细胞裂解物或分离的线粒体结合，提示通过伴侣蛋白介导的载体途径运输。SLC4A11 与 HSP90 或 HSC70 复合物直接结合在线粒体表面受体 TOM70 上。干扰这种运输会导致功能性线粒体谷氨酰胺分解代谢和活性氧产生增加。此外，谷氨酰胺（Gln）的使用增加了 HCECs 的全细胞裂解物或 HA-SLC4A11 转染的成纤维细胞中纯化的线粒体中的 SLC4A11、HSP70 和 HSP90 的表达。
HSP90 和 HSC70 对于角膜内皮细胞和肾脏中线粒体 SLC4A11 的转运至关重要。Gln 促进 SLC4A11 向线粒体的输入，而 Gln 分解代谢产生的持续氧化应激诱导 HSP70 和 HSP90，从而保护细胞免受氧化应激。
