Modifiers of prion protein biogenesis and recycling identified by a highly parallel endocytosis kinetics assay.
The cellular prion protein, PrPC, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrPScis the causative agent of prion diseases. Conversion of PrPCinto PrPScis thought to take place at the cell surface or in endolysosomal organelles. Understanding the intracellular trafficking of PrPCmay, 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 PrPCThe assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrPCtrafficking. Simultaneous administration of FRET donor and acceptor anti-PrPCantibodies to living cells yielded a measure of PrPCsurface density, whereas sequential addition of each antibody visualized the internalization rate of PrPC(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 PrPCinternalization, whereas down-regulation of GIT2 and VPS28 increased PrPCinternalization. PrPCcell-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 PrPCtrafficking and demonstrate the power of this assay for identifying modulators of PrPCtrafficking.
Institute of Neuropathology, University of Zurich, CH-8091 Zurich, Switzerland.
Boris A
BA
Ballmer
Institute of Neuropathology, University of Zurich, CH-8091 Zurich, Switzerland.
Rita
R
Moos
Institute of Molecular Life Sciences, University of Zurich, CH-8091 Zurich, Switzerland; Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland.
Prisca
P
Liberali
Institute of Molecular Life Sciences, University of Zurich, CH-8091 Zurich, Switzerland.
Lucas
L
Pelkmans
Institute of Neuropathology, University of Zurich, CH-8091 Zurich, Switzerland. Electronic address: simone.hornemann@usz.ch.
Simone
S
Hornemann
Institute of Neuropathology, University of Zurich, CH-8091 Zurich, Switzerland. Electronic address: adriano.aguzzi@usz.ch.
Adriano
A
Aguzzi
D000072283:A549 Cells; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D018621:PrPC Proteins / Q000235:genetics / Q000378:metabolism*; D018620:PrPSc Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D017527:Receptor, IGF Type 2 / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
C120700:EHD1 protein, human; D056827:Endosomal Sorting Complexes Required for Transport; D018621:PrPC Proteins; D018620:PrPSc Proteins; D017527:Receptor, IGF Type 2; C476409:VPS28 protein, human; C419573:VPS35 protein, human; D033921:Vesicular Transport Proteins; C034304:cation-dependent mannose-6-phosphate receptor; C427755:RAB5C protein, human; D020696:rab5 GTP-Binding Proteins
cell surface; endocytosis; fluorescence resonance energy transfer (FRET); high-throughput screening (HTS); prion disease