Vaz, M. | Search bioRxiv

bioRxiv2026

Molecular basis of Salla Disease: R39C Mutation Effects on the Lysosomal Transporter Sialin

Salla disease is caused by a genetic mutation in sialin, a lysosomal membrane transporter, which exports sialic acid from lysosomes. Substrate translocation occurs via a rocker-switch mechanism that alternately exposes the substrate-binding site to the lysosomal lumen and the cytosol. The pathogenic mutation R39C found in most Salla disease patients decreases the lysosomal localisation and the transport activity. In this study, we used computational and mutagenesis approaches to elucidate the molecular effects of the R39C mutation. Using three-dimensional models of human sialin in the lumen-open (LO) and cytosol-open (CO) states combined with the mutagenesis of selected residues, we identify a critical "triplet" motif comprising R39, E194, and E262, which is associated with an ionic lock formed between K197 and D350 in the LO conformation. Molecular dynamics simulations suggest that the electrostatic triplet negatively modulates the ionic lock, and are consistent with a strengthened ionic lock in R39C sialin, potentially favouring the LO state. To assess the global effects of the R39C mutation, we computed dynamic cross-correlation matrices and identified correlation patterns consistent with an allosteric coupling between the ionic lock K197/D350 and the region surrounding the sialic acid binding site in wild-type sialin, whereas in the LO state of R39C sialin, this communication preferentially bypasses this region. Therefore, the R39C mutation may impede the LO to CO conformational transition required for sialic acid transport, providing a plausible mechanistic framework for the decreased transport activity, and possibly the decreased lysosomal localisation, observed in Salla disease. HighlightsO_LIThe R39 residue participates in an interaction triplet, which negatively regulates an ionic lock stabilising the lumen-open conformation C_LIO_LIThe R39C mutation is associated with a stronger ionic lock in the simulations, and may favour the lumen-open state C_LIO_LICorrelation network analysis suggests an allosteric coupling between the ionic lock and the region surrounding the sialic acid binding site C_LIO_LIThe R39C mutation alters the inferred allosteric coupling between the ionic lock and the region surrounding the sialic acid binding site C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/719580v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1bf7144org.highwire.dtl.DTLVardef@1a53ab8org.highwire.dtl.DTLVardef@b2249forg.highwire.dtl.DTLVardef@1827244_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Mitotic checkpoint protein Mad1 is required for early Nup153 recruitment to chromatin and nuclear envelope integrity

The nucleoporin Nup153 is a multifunctional protein and the mitotic checkpoint protein Mad1one of its many binding partners. The functional relevance of their interaction has remained elusive. Here, we have further dissected Nup153s and Mad1s interface and functional interplay. By in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is prerequisite for the Nup153-Mad1 interaction. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of the membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between the inner and outer nuclear membranes. Nup153 depletion, but not of Mad1, caused defects in interphase NPC assembly with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Together our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE reformation in concert with Mad1 and in interphase NPC assembly, independent of Mad1. SummaryThe mitotic checkpoint protein is required for Nup153 recruitment to anaphase chromatin and in turn post-mitotic, but not interphase nuclear pore complex assembly.

cell biology↗