Modifiers of prion protein biogenesis and recycling identified by a highly parallel endocytosis kinetics assay.

The cellular prion protein, PrP, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrP is the causative agent of prion diseases. Conversion of PrP into PrP is thought to take place at the cell surface or in endolysosomal organelles. Understanding the intracellular trafficking of PrP may, therefore, help elucidate the conversion process. Here we describe a time-resolved fluorescence energy transfer (FRET) assay reporting membrane expression and real-time internalization rates of PrP The assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrP trafficking. Simultaneous administration of FRET donor and acceptor anti-PrP antibodies to living cells yielded a measure of PrP surface density, whereas sequential addition of each antibody visualized the internalization rate of PrP (Z' factor >0.5). RNA interference assays showed that suppression of AP2M1 (AP-2 adaptor protein), RAB5A, VPS35 (vacuolar protein sorting 35 homolog), and M6PR (mannose 6-phosphate receptor) blocked PrP internalization, whereas down-regulation of GIT2 and VPS28 increased PrP internalization. PrP cell-surface expression was reduced by down-regulation of RAB5A, VPS28, and VPS35 and enhanced by silencing EHD1. These data identify a network of proteins implicated in PrP trafficking and demonstrate the power of this assay for identifying modulators of PrP trafficking.