Homeostasis of SLC4A11 protein is mediated by endoplasmic reticulum-associated degradation

The cornea is an important tissue that refracts light, and the corneal endothelium prevents edema of the corneal stroma by acting as a barrier and a pump for the transport of essential molecules/ions. Sodium bicarbonate transporter-like protein 11 (SLC4A11) is a transporter present in the corneal endothelium, and its mutation causes corneal endothelial disease. Here, we aimed to investigate the degradation pathway of SLC4A11. Quantitative PCR analysis revealed that two variants of SLC4A11 transcripts, variant 2 (SLC4A11-B) and variant 3 (SLC4A11-C), were expressed in human corneal endothelial tissues. Transient overexpression of these variants in HEK293T cells revealed that SLC4A11-B abundantly localized to the cell membrane. Furthermore, SLC4A11-B-transfected HEK293T cells expressed the mature glycosylated forms and immature non-glycosylated forms of SLC4A11. Cycloheximide chase experiments revealed that mature SLC4A11 showed high degradation stability; however, degradation of immature SLC4A11-B was significantly faster than that of immature SLC4A11-C. Therefore, we performed further degradation analysis of the SLC4A11 mutants, which are classified into ER-retained and cell surface-associated mutants similar to the wild type. Compared to the wild type, ER-retained mutants S213P and W240P showed delayed degradation but the cell surface-associated mutants showed minimal degradation. Further analysis using proteasome inhibitors revealed that degradation of immature SLC4A11 was delayed after treatment with the proteasome inhibitors, MG-132 and bortezomib, and was mediated by poly-ubiquitination. Moreover, the degradation of immature SLC4A11 protein was suppressed by Eeyarestatin I, an ER-associated protein degradation (ERAD) inhibitor. Collectively, these data suggest that SLC4A11 protein is degraded via ERAD. •The proteolysis of immature SLC4A11 protein differs between isoforms.•Immature SLC4A11 proteins and some ER-retained mutants show delayed proteolysis.•Immature SLC4A11 protein is degraded by ERAD via proteasome.