The cytoplasmic domain is essential for transport function of the integral membrane transport protein SLC4A11

Abstract

Large cytoplasmic domains (CD) are a common feature among integral membrane proteins. In virtually all cases, these CD have a function (e.g., binding cytoskeleton or regulatory factors) separate from that of the membrane domain (MD). Strong associations between CD and MD are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, the mutations of which cause genetic corneal blindness. SLC4A11 has a 41-kDa CD and a 57-kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK-293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that the structure of CD-SLC4A11 is similar to that of the Cl(-)/HCO3(-) exchange protein AE1 (SLC4A1) CD. Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that the structure of CD-AE1 is similar to that of CD-SLC4A11. The CD-AE1-MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensable role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD-SLC4A11 and MD-SLC4A11, these domains specifically associate when coexpressed in HEK-293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the CD has strong associations with the integral MD, which contributes to membrane transport function.

SLC4A11 Three-Dimensional Homology Model Rationalizes Corneal Dystrophy-Causing Mutations

“…Although SLC4All and Band 3 are both members of the SLC4 family, they differ in their transport substrates and in the interaction of their cytoplasmic and membrane domains [Loganathan et al., ]. We thus analyzed amino acid sequence conservation in Band 3 and SLC4A11 across the membrane domains of 30 species, including fish, birds, and mammals, using the program ConSurf [Glaser et al., ; Landau et al., ; Celniker et al., ] (Suppl.…”

“…Band 3 and SLC4A11 have N‐terminal cytoplasmic domains and C‐terminal membrane domains of similar size. The cytosolic domain of SLC4A11 contains approximately one‐third of reported corneal dystrophy‐causing mutations, and is essential for transport and overall stability of SLC4A11 [Loganathan et al., ]. Recently the cytoplasmic and membrane domains of SLC4A11 were suggested to closely associate on the basis of: (1) An inability for either domain to accumulate when heterologously expressed alone in a tissue culture model, (2) An association between the two domains when co‐expressed, and (3) The catalytic defect found in the cytoplasmic domain mutation, p.Arg125His, which suggested a role of the cytoplasmic domain in transport mechanism [Loganathan et al., ].…”

Molecular phenotype of SLC4A11 missense mutants: Setting the stage for personalized medicine in corneal dystrophies

“…Strikingly, intracellular‐retained mutants are enriched in the plane of the lipid bilayer (20 out of 27), whereas outside the bilayer only six out of 22 cause ER retention (Figure A). A structural homology model for SLC4A11 cytoplasmic domain (Loganathan, Lukowski, & Casey, ) based on the SLC4A1 cytoplasmic domain structure (Zhang, Kiyatkin, Bolin, & Low, ), illustrates that in the densely packed center of the cytoplasmic domain, the protein may be more sensitive to misfolding, leading to recognition by ER quality control mechanisms and ER retention (Figure B and C).…”

“…Mutations in the cytoplasmic domain (Figure B and C, residues 1–374) are difficult to assess as no role for this domain has been described. However, the cytoplasmic domain has strong interactions with the membrane domain and may contribute in some way to transmembrane substrate translocation (Loganathan et al., ). This is underscored by the observation that only seven out of 19 mutations in the cytoplasmic domain result in intracellular retention of mutants, suggesting that most cytoplasmic domain mutations impair some necessary function of SLC4A11.…”

“…This is underscored by the observation that only seven out of 19 mutations in the cytoplasmic domain result in intracellular retention of mutants, suggesting that most cytoplasmic domain mutations impair some necessary function of SLC4A11. Beyond a possible role in substrate funneling to the membrane domain (Loganathan et al., ), perhaps the cytoplasmic domain interacts with cytoplasmic proteins. Other members of the SLC4 family, best illustrated by SLC4A1 (AE1) (Alka & Casey, ), have cytoplasmic domains that interact with cytoskeleton.…”