Biology subjects

Mindell, J. A.

Publications and source records attributed to Mindell, J. A..

5 recordsLinked to original sources

bioRxiv2025

Mechanism of phosphoinositide regulation of lysosomal pH via inhibition of CLC-7

AbstractLysosomes process cellular waste and coordinate responses to metabolic challenge. Central to lysosomal homeostasis are phosphoinositide lipids, key signaling molecules which establish organelle identity, regulate membrane dynamics and are tightly linked to the pathophysiology and therapy of lysosomal storage disorders, neurodegeneration, and cancer. Phosphatidylinositol 3,5- bisphosphate (PI(3,5)P2) interacts with multiple lysosomal membrane proteins and plays a critical role in regulating lysosomal pH by directly inhibiting the chloride/proton antiporter ClC-7, though the molecular mechanism of this inhibition remains unclear. Here, using a combination of functional, structural, and computational analysis, we demonstrate that PI(3,5)P2 binding dramatically remodels the structure of ClC-7 by inducing close association between cytosolic and transmembrane domains. Disease-causing mutations show increased transport activity through loss of PI(3,5)P2 binding and subsequent inhibition. Conversely, ClC-7 activation is correlated with dissociation and increased disorder of the cytoplasmic domain along with novel transmembrane domain conformations, revealing a mechanistic link between specific lysosomal lipids, transporter regulation, and the enigmatic basis of the ClC-7 slow gate.

biochemistry↗

bioRxiv2023

Exploring the K+ binding site and its coupling to transport in the neurotransmitter:sodium symporter LeuT

The neurotransmitter:sodium symporters (NSSs) are secondary active transporters that couple the reuptake of substrate to the symport of one or two sodium ions. One bound Na+ (Na1) contributes to the substrate binding, while the other Na+ (Na2) is thought to be involved in the conformational transition of the NSS. Two NSS members, the serotonin transporter (SERT) and the Drosophila dopamine transporter (dDAT), also couple substrate uptake to the antiport of K+ by a largely undefined mechanism. We have previously shown that the bacterial NSS homologue, LeuT, also binds K+, and could therefore serve as a model protein for the exploration of K+ binding in NSS proteins. Here, we characterize the impact of K+ on substrate affinity and transport as well as on LeuT conformational equilibrium states. Both radioligand binding assays and transition metal ion FRET (tmFRET) yielded similar K+ affinities for LeuT. K+ binding was specific and saturable. LeuT reconstituted into proteoliposomes showed that intra-vesicular K+ dose-dependently increased the transport velocity of [3H]alanine, whereas extra-vesicular K+ had no apparent effect. K+-binding induced a LeuT conformation distinct from the Na+- and substrate-bound conformation. Conservative mutations of the Na1 site residues affected the binding of Na+ and K+ to different degrees. The Na1 site mutation N27Q caused a >10-fold decrease in K+ affinity but at the same time a [~]3-fold increase in Na+ affinity. Together, the results suggest that K+-binding to LeuT modulates substrate transport and that the K+ affinity and selectivity for LeuT is sensitive to mutations in the Na1 site, pointing toward the Na1 site as a candidate site for facilitating the interaction with K+ in some NNSs.

biophysics↗

bioRxiv2022

Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter

VcINDY, the sodium-dependent dicarboxylate transporter from Vibrio cholerae, is responsible for C4- and C5-carboxylate uptake into cells. The molecular mechanism of how VcINDY physically moves substrates across the membrane, and does so in an energetically efficient manner, is unclear. Here, we use single-molecule fluorescence resonance energy transfer experiments to directly observe the individual mechanistic steps that VcINDY takes to translocate substrates across a lipid bilayer, and then test key predictions of transport cycle mechanistic models. Our data provide the first direct evidence that VcINDY undergoes stochastic, elevator-type conformational motions that enable substrate translocation. Kinetic analysis suggests that the two protomers of the VcINDY homodimer undergo those motions in a non-cooperative manner, and thus catalyze two independent transport reactions. The relative substrate independence of those motions supports the notion that the VcINDY transport cycle maintains strict co-substrate coupling using a cooperative binding mechanism. Finally, thermodynamic modeling provides insight into how such a cooperative binding mechanism provides a generalized approach to optimizing transport for many secondary active transporters. Significance StatementTransporter proteins use energy to move molecular materials into and out of cells. To be efficient, the transporter motions responsible for moving the molecules must be tightly choreographed to avoid wasting energy without transporting anything. By measuring the motions and kinetics of a prototypical transporter (VcINDY) at the single-molecule level, this study finds the first evidence that transporters like VcINDY achieve efficient transport by coordinating constantly dynamic, "elevator-type" motions while sitting in the cellular membrane. The efficiency of these surprisingly dynamic transporters is then revealed by thermodynamic modeling, which explains the molecular basis behind how highly cooperative, substrate binding reactions may have evolved as the optimal strategy for maximizing transporter efficiency.

biophysics↗

bioRxiv2021

Tonic inhibition of the chloride/proton antiporter ClC-7 by PI(3,5)P2 is crucial for lysosomal pH maintenance

The acidic luminal pH of lysosomes, maintained within a narrow range, is essential for proper degrative function of the organelle and is generated by the action of a V-type H+ ATPase, but other pathways for ion movement are required to dissipate the voltage generated by this process. ClC-7, a Cl-/H+ antiporter responsible for lysosomal Cl- permeability, is a candidate to contribute to the acidification process as part of this "counterion pathway". The signaling lipid PI(3,5)P2 modulates lysosomal dynamics, including by regulating lysosomal ion channels, raising the possibility that it could contribute to lysosomal pH regulation. Here we demonstrate that depleting PI(3,5)P2 by inhibiting the PIKfyve kinase causes lysosomal hyperacidification, primarily via an effect on ClC-7. We further show that PI(3,5)P2 directly inhibits ClC-7 transport and that this inhibition is eliminated in a disease-causing gain-of-function ClC-7 mutation. Together these observations suggest an intimate role for ClC-7 in lysosomal pH regulation.

biophysics↗

bioRxiv2020

Substrate-dependent accessibility changes in the Na+-dependent C4-dicarboxylate transporter, VcINDY, suggest differential substrate effects in a multistep mechanism.

Members of the divalent anion sodium symporter (DASS) family (SLC13 in humans) play critical roles in metabolic homeostasis, influencing many processes including fatty acid synthesis, insulin resistance, adiposity, and lifespan determination. DASS transporters catalyse the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and VcINDY conformation using a site-specific alkylation strategy to probe the solvent accessibility of several broadly distributed positions in VcINDY in the presence and absence of substrates (Na+ and succinate). Our findings reveal that accessibility to all positions tested can be modulated by the presence of substrates, with the majority becoming less accessible in the presence of Na+. Mapping these solvent accessibility changes onto the known structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified intermediate state. We also observe substantial, separable effects of Na+ and succinate binding at several amino acid positions suggesting distinct effects of the two substrates. Furthermore, analysis of a solely succinate-sensitive residue indicates that VcINDY binds its substrate with a low affinity and proceeds via an ordered process in which one or more Na+ ions must bind prior to succinate. These findings provide insight into the mechanism of VcINDY, which is currently the only structural-characterised representative of the entire DASS family.

biochemistry↗