Dynamic microscopy: Reconstructing a novel lysosomal trafficking pathway

Abstract

The delivery of newly synthesized lysosomal proteins to endosomes and lysosomes is dependent on several functionally distinct compartments, i.e., the endoplasmic reticulum (ER), the Golgi apparatus and small carrier vesicles. These compartments also play a role in the synthesis, post-translational modification, sorting and distribution of proteins to other destinations. In fact, most cargo is sorted within, and exits from, the trans-Golgi network (TGN). Proteins delivered to the endosomal/lysosomal system include a large and diverse class of hydrolytic enzymes and non-enzymatic activator proteins that were thought to be directed to the lysosomes by their binding to mannose-6-phosphate receptors (MPRs). Surprisingly, in I-cell disease, in which the MPR pathway is disrupted, the non-enzymatic sphingolipid activator protein, prosaposin, continues to traffic to lysosomes. The use of dynamic immunoconfocal and immunoelectron microscopy along with mutational analysis and chimeric protein technology led us to the discovery of a new lysosomal sorting receptor, sortilin. We found that prosaposin is targeted to the lysosomes through an interaction with sortilin. Deletion of the C-terminus of prosaposin abolished its transport to the lysosomes, and mutational analysis revealed that the first half of the prosaposin C-terminus (aa524-540) contains a motif required for its binding to sortilin and its transport to lysosomes. Additionally, a chimeric construct consisting of albumin and a distal segment of prosaposin, which includes its C-terminus, resulted in the routing of albumin to lysosomes. In conclusion, we have identified a specific region in the C-terminus of prosaposin, as well as amino acid residues that are critical for the binding of prosaposin to sortilin and its subsequent lysosomal trafficking.