Membrane Domain Localization and Interaction of the Prion-Family Proteins, Prion and Shadoo with Calnexin

The cellular prion protein (PrP^C) is renowned for its infectious conformational isoform PrP^Sc, capable of templating subsequent conversions of healthy PrP^Cs and thus triggering the group of incurable diseases known as transmissible spongiform encephalopathies. Besides this mechanism not being fully uncovered, the protein's physiological role is also elusive. PrP^C and its newest, less understood paralog Shadoo are glycosylphosphatidylinositol-anchored proteins highly expressed in the central nervous system. While they share some attributes and neuroprotective actions, opposing roles have also been reported for the two; however, the amount of data about their exact functions is lacking. Protein-protein interactions and membrane microdomain localizations are key determinants of protein function. Accurate identification of these functions for a membrane protein, however, can become biased due to interactions occurring during sample processing. To avoid such artifacts, we apply a non-detergent-based membrane-fractionation approach to study the prion protein and Shadoo. We show that the two proteins occupy similarly raft and non-raft membrane fractions when expressed in N2a cells and that both proteins pull down the chaperone calnexin in both rafts and non-rafts. These indicate their possible binding to calnexin in both types of membrane domains, which might be a necessary requisite to aid the inherently unstable native conformation during their lifetime.