Structures and Mechanisms of Lysosomal Transporters

Abstract

Lysosomal membrane transporters are indispensable for maintaining lysosomal homeostasis and proper function. Indeed, mutations in these key proteins can lead to debilitating disorders known as lysosomal storage diseases. Cystinosin and Sialin are two such transporters. Both proteins utilize the low pH environment to transport their main substrates of interest from the lumen to the cytosol where they can be reused by the cell. Understanding how these proteins work at an atomic level can help us understand their overall function, the roles they play in lysosomal signaling pathways, and may enable the future development of therapeutics to treat their associated disorders. Mutations in Cystinosin cause Cystinosis, a neurodegenerative disorder that occurs when Cystinosin's substrate, the dimeric form of cysteine called cystine, builds up in the lysosome. While there currently is a treatment for this disorder, how mutations disrupt this transport and how the protein utilizes the proton gradient remain a mystery. Similarly, Sialin causes a variety of free sialic acid storage diseases that result in developmental delays as well as neurodegeneration. Unlike Cystinosis, there is currently no approved treatment for these disorders. Like Cystinosin, the structure and mechanism of transport remain unknown. The work presented herein reveals the cryo-EM structures of Cystinosin and Sialin at near atomic level resolution. These structures, captured in both cytosol- and lumen-open conformations as well as substrate bound states, reveal not only the mechanisms of conformational changes but also the residues involved in the substrate binding pocket(s). Along with the accompanying functional assays, I demonstrate that the majority of disease-causing mutations in both Cystinosin and Sialin center around their ability to bind their respective substrates. Additionally, I reveal the potential proton sensors of both proteins involved in their substrate symport. In the case of Sialin, I also hypothesize that its proton sensor could potentially double as a membrane potential sensor for its transport of neurotransmitters into synaptic vesicles. This work paves the way for understanding the greater PQ-loop family (Cystinosin) and SLC17 family (Sialin) of transporters. It also builds a foundation upon which future therapeutics can be designed to treat their associated disorders