Abstract
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.
Highlights
The lysosome after they are released during degradation of glycolipids and glycoproteins
Site-directed mutagenesis of VGLUT2 driven by this model has been used to demonstrate that a His in TM2 that is specific to the VGLUTs and Arg and Glu residues in TM4 that are conserved in the SLC17 family are required for transport activity [10]
7.0, NANA reduced the inhibitory effect, but glucuronic acid (GlcUA) failed to sonable approximation
Summary
Reagents—Methanethiosulfonate-ethyltrimethylammonium (MTSET) was obtained from Anatrace (Maumee, OH) and N-ethylmaleimide (NEM) from Sigma-Aldrich. 3H-NANA (20 Ci/mmol) and 3H-GlcUA (10 Ci/mmol) were obtained from American Radiolabeled Chemicals Sulfhydryl-reactive Reagent Accessibility—For NEM inhibition assays, cells were washed once with Krebs-Ringer-HEPES (KRH), pH 7.5, and incubated in the same buffer supplemented with NEM at a concentration of 2 mM for 5 min at 23 °C. Reactions were stopped by adding 4 volumes of KRH, pH 7.5, and the cells were washed once more with the same buffer. To test the effects of substrates or pH, cells were incubated in KRM, pH 5.5, or KRH, pH 7.0 or pH 8.5, with or without substrates at indicated concentrations for 8 min at 23 °C. Sulfhydryl reagents were added to the final indicated concentration, and incubation was continued for 2 min. The reaction was stopped by dilution, the cells were washed once, and uptake was measured as indicated above. Calculations for p values were done using two-tailed unpaired t test or one-way analysis of variance with Bonferroni correction as appropriate
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