title: 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.
authors.affiliation: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Sampath K
authors.initials: SK
authors.lastname: Loganathan
authors.affiliation: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Chris M
authors.initials: CM
authors.lastname: Lukowski
authors.affiliation: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada joe.casey@ualberta.ca.
authors.forename: Joseph R
authors.identifier: http://orcid.org/0000-0002-9890-5056
authors.initials: JR
authors.lastname: Casey
mesh_terms: D027321:Anion Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism*; D001639:Bicarbonates / Q000737:chemistry* / Q000378:metabolism*; D001693:Biological Transport, Active / Q000502:physiology; D003593:Cytoplasm / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D027321:Anion Transport Proteins; D017920:Antiporters; D001639:Bicarbonates; C425809:SLC4A11 protein, human
keywords: SLC4A11; corneal dystrophy; cytoplasmic domain; membrane domain; protein misfolding