Small Molecule and Lipid Binding Mechanisms of Sortilin and Vps10 Indicated in Altered Trafficking Patterns and Membrane Fusion

Abstract

This study focuses on the regulation of sortilin/Vps10p‐directed protein trafficking. Human sortilin is a Vps10 domain‐containing transmembrane receptor that interacts with Apo B100‐containing VLDL, PCSK9 and LDL in the lumen of hepatic secretory vesicles. The Vps10 domain name comes from the yeast orthologue Vps10p, a carboxypeptidase Y (CPY) sorting receptor responsible for trafficking proteins to the vacuole in yeast. We have recently shown that vesicular VLDL contains phosphatidylinositol (3,4,5)‐trisphosphate (PIP3). Furthermore, we found that sortilin directly binds PIP3 using liposome binding assays as well as surface plasmon resonance using nanodiscs. We found that small molecules designed to bind human sortilin were also effective in yeast, leading to altered protein sorting to the yeast vacuole and secretion of CPY. Additional efforts using liposome floatation assays and microscale thermophoresis show that Vps10p and sortilin bind to additional membrane lipids. We hypothesize that differential binding different types of membrane lipids regulates Vps10‐controlled protein sorting. Further characterization of sortilin probes in yeast identified a variety of potential protein targets including the Rab7 orthologue Ypt7. This late endosomal GTPase is required for retrograde trafficking to the Golgi, anterograde traffic to the endolysosomal pathway, and vacuole homotypic fusion. Both deletion of VPS10 or binding of the receptor with sortilin‐targeting compounds led to defective Ypt7 trafficking to vacuoles and inhibited homotypic vacuole fusion. To complement direct binding studies, yeast Vps10p homologues have been identified and structural research performed to better understand binding features of Vps10‐like luminal domains. Elucidation of the relationship between specific luminal binding interactions of sorting receptors and vesicle trafficking predestination should allow better understanding of mechanistic interactions of yeast Vps10p with cytosolic trafficking factors and components.Support or Funding InformationNIGMS R01‐GM101132