Methods and Resources
Modifiers of prion protein biogenesis and recycling identified by a highly parallel endocytosis kinetics assayPrPC membrane expression and internalization rates

https://doi.org/10.1074/jbc.M116.773283Get rights and content
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The cellular prion protein, PrPC, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrPSc is the causative agent of prion diseases. Conversion of PrPC into PrPSc is thought to take place at the cell surface or in endolysosomal organelles. Understanding the intracellular trafficking of PrPC 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 PrPC. The assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrPC trafficking. Simultaneous administration of FRET donor and acceptor anti-PrPC antibodies to living cells yielded a measure of PrPC surface 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 PrPC internalization, whereas down-regulation of GIT2 and VPS28 increased PrPC internalization. PrPC 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 PrPC trafficking and demonstrate the power of this assay for identifying modulators of PrPC trafficking.

cell surface
endocytosis
fluorescence resonance energy transfer (FRET)
high-throughput screening (HTS)
prion disease

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This article contains supplemental Figs. S1–S6.

The authors declare that they have no conflicts of interest with the contents of this article.

Recipient of the Swiss Initiative in Systems Biology, SystemsX.ch (SynucleiX) and the commission innovations of the University Hospital of Zurich.

Recipient of an Advanced Grant of the European Research Council, a European Union Framework 7 Grant (NEURINOX), the Swiss National Foundation, the Clinical Research Priority Programs “Small RNAs” and “Human Hemato-Lymphatic Diseases,” the Swiss Initiative in Systems Biology, SystemsX.ch (PrionX, SynucleiX), and the Gelu Foundation.

1

Recipient of the Swiss National Science Foundation (PP00P3_157531).

2

Recipient of a Consolidator Grant of the Swiss National Science Foundation, the University of Zurich Research Priority Program in Systems Biology, and the Swiss Initiative in Systems Biology, SystemsX.ch (LipidX, PrionX).