A major unresolved question is where PrPc-PrPSc transconformation occurs. Both PrPc and PrPSc have been localized at the plasma membrane and have been shown to undergo endocytosis [46,47], so it is likely that the plasma membrane and/or the endolysosomal compartment participate in PrPSc formation. In addition, in the case of inherited diseases, pulse-chase experiments indicate that the pathological conversion of mutant PrP to the PrPSc-like conformer proceeds in a stepwise manner, via a series of identifiable biochemical intermediates, and that one of the earliest properties of PrPSc (i.e., resistance to cleavage by phosphatidylinositol phospholipase C) is acquired in the endoplasmic reticulum (ER) . Moreover, the ER could also be implicated in the degradation of misfolded PrP mutants via the ER-associated degradation (ERAD) pathway [48-50].
Hence, the available data indicate that in the case of genetic prion diseases originating from PrP mutants (for a review, see ), the ER might be directly involved in protein transconformation and consequent PrPSc formation. Conversely, because in infected cells the stimulation of PrPc retrograde transport results in an increase in the PrPSc form , we recently proposed  that in infectious diseases the ER could represent an amplification compartment for PrPSc produced earlier at other subcellular sites.
Detergent-resistant microdomains in the ER (ER-rafts) could also be involved in the folding of PrPc . Like neuronal cell lines, epithelial cells synthesize four different isoforms of PrPc corresponding to the unglycosylated, monoglycosylated, immature diglycosylated, and mature diglycosylated isoforms. Interestingly, it was found that the immature diglycosylated precursor form of PrPc was recovered in a DRM fraction in the ER. Using cholesterol- and sphingolipid-depleting drugs in concomitance with pulse-chase experiments, we were able to show that this earlier raft association was crucial for the correct folding of PrP. Indeed, a portion of the protein became misfolded in the ER when cholesterol was extracted .
The mechanism that underlies the specificity of cholesterol depletion for PrPc misfolding could be explained in two ways:
• either cholesterol depletion could perturb the formation of specific DRMs present in the ER, leading to an interference of the association of the immature PrPc isoform with rafts; or
• cholesterol itself could be directly involved in the folding of PrPc by acting as a lipochaperone in the ER [53,54].
In contrast with the proposed role for the ER in prion conversion, the results of several studies have indicated that the conversion of PrPc to a protease- and phos-pholipase-resistant state is an event that occurs after the protein has reached the cell surface [47,55,56]. Hence, the release of PrPc from the cell surface by different methods [47,55,57] and exposure of PrPc to different antibodies  prevents PrPSc formation in infected cells.
Therefore, the current evidence suggests that both the ER and the plasma membrane might be important sites for prion formation, and that they are differently involved in the genetic and infectious diseases. Whilst in the case of genetic prion diseases, the ER could be involved in the generation of PrPSc or PrPSc-like con-formers from PrP mutants , in the case of infectious diseases, the first contact between physiological and pathological PrP isoforms could occur at the plasma membrane, although the transconformation might occur later after internaliza-tion.
After its transport to the plasma membrane, PrPc is constitutively internalized and recycles back to the surface [31,46]. The endocytic recycling pathway is of interest from the standpoint of prion generation, since there is evidence that initial steps in the conversion of PrPc into PrPSc may take place on the plasma membrane or following the internalization of PrPc [47,51,55]. The route and mechanism of internalization of PrPc are controversial because both caveolae and clathrin-coated pits have been shown to be involved (for a review, see [31,46]). Clathrin-coated pits appear to be primarily responsible for endocytic uptake of PrPc. This conclusion is based on immunogold localization of PrPc in these organelles by electron micros copy [58,59] and inhibition of PrPc internalization by incubating cells in hypertonic sucrose, which disrupts clathrin lattices .
Because PrPc lacks a cytoplasmic tail that could interact directly with adaptor proteins and clathrin , several candidate proteins have been proposed to be PrP-interacting partners mediating its internalization via the clathrin pathway. Specifically, a basic amino acid motif found in the N-terminal region of PrPc [31,60,61] has been shown to be essential for coated pit localization and internalization. In contrast to these studies, the presence of PrPc in caveolar-like domains (CLDs) has also been extensively reported [62,63]. In Chinese hamster ovary (CHO) cells, which express caveolin-1, PrPc is enriched in caveolae both at the TGN and at the plasma membrane and in interconnecting chains of endocytic caveolae, but it is apparently absent in clathrin-coated pits and vesicles .
The initial recruitment of PrPc to pre-endocytic membranes may therefore be a complex event which occurs by more than one mechanism. It is possible that PrPc is internalized by default by clathrin-coated vesicles, and that caveolae or CLDs provide alternative internalization pathways occurring in particular cells or conditions.
These different mechanisms may provide a range of possibilities for protein conversion and pathological spreading. Indeed, both CLDs and clathrin-coated pits have been suggested to be involved in PrPc to PrPSc transconformation [60,63], but until the internalization pathway of PrPc is clarified it will be difficult to establish the involvement of one or the other pathway in transconformation.
The fact that most PrPc molecules reside in raft domains does not argue against an association of the protein with coated-pits, because only a small fraction of the PrPc molecules are undergoing endocytosis at any one time, and this fraction has probably left the raft domain to enter the coated pits. In this context, it has been demonstrated  that PrPc, prior to endocytosis, leaves the detergent-insoluble raft environment to cluster, along with TfR (the prototypical transmembrane protein endocytosed by coated pits and excluded by lipid rafts)  in non-raft membranes. Thus, PrPc on the cell surface rapidly traffics through two very different membrane environments, probably with different consequences for its conformational stability.
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