Structure-function studies of the SLC17 transporter sialin identify crucial residues and substrate-induced conformational changes.
Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H(+)-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members.
Courville
Pascal
P
Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California 94305, USA.
Quick
Matthias
M
Reimer
Richard J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D020723:Glucuronic Acid / Q000737:chemistry; D006801:Humans; D016464:Lysosomal Storage Diseases / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D027361:Organic Anion Transporters / Q000737:chemistry*; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D027981:Symporters / Q000737:chemistry*
D027361:Organic Anion Transporters; D020033:Protein Isoforms; D027981:Symporters; C421224:sialic acid transport proteins; D020723:Glucuronic Acid; D019158:N-Acetylneuraminic Acid; D003545:Cysteine