PrP-containing aggresomes are cytosolic components of an ER quality control mechanism

Abstract

Limited detoxification capacity often directs aggregation-prone, potentially hazardous, misfolded proteins to be deposited in designated cytosolic compartments known as 'aggresomes'. The roles of aggresomes as cellular quality control centers, and the cellular origin of the deposits contained within these structures, remain to be characterized. Here, we utilized the observation that the prion protein (PrP, also known as PRNP) accumulates in aggresomes following the inhibition of folding chaperones, members of the cyclophilin family, to address these questions. We found that misfolded PrP molecules must pass through the endoplasmic reticulum (ER) in order to be deposited in aggresomes, that the Golgi plays no role in this process and that cytosolic PrP species are not deposited in pre-existing aggresomes. Prior to their deposition in the aggresome, PrP molecules lose the ER localization signal and have to acquire a GPI anchor. Our discoveries indicate that PrP aggresomes are cytosolic overflow deposition centers for the ER quality control mechanisms and highlight the importance of these structures for the maintenance of protein homeostasis within the ER.

Vesicle‐mediated secretion of misfolded prion protein molecules from cyclosporin A‐treated cells

“…pDsRed2-ER vector (632409; Clontech Laboratories, Mountain View, CA, USA) was used to label the ER fluorescently. The PrP constructs used have been previously described (17). Rab27B shRNA plasmids were isolated from glycerol stock by using Qiagen (Hilden, Germany) Plasmid Plus Midi Kit (12943).…”

“…PrP-containing exosomes originate from the ER Misfolded PrP molecules that are deposited in aggresomes originate from the ER (17); however, a fraction of the molecules that are expressed in PrP overexpressing cells fail to enter the ER, stay entirely cytosolic, and are directed for degradation by the proteasome (32). Although CsA was shown to induce the misfolding of PrP by inhibiting the luminal chaperone cyclophilin B (17), it is possible that CsA also affects the metabolism of cytosolic PrP by inhibiting the activity of other cyclophilins. Thus, we asked whether CsA-induced exosomes contained PrP molecules that pass through the ER or whether they secrete PrP species, which fail to enter that organelle.…”

“…To address that, we transiently transfected N2a cells with either 1 of 2 plasmids: one that carries PrP that lacks the ER localization signal and thus, remains entirely cytosolic (DSP-PrP), or a second plasmid that expresses PrP that bears the highly efficient ER localization signal of the hormone prolactin (Prl). The Prl signal has been reported to mediate the entry of PrP into ER-like vesicles in an in vitro system (33) and into the ER of cells (17). The cells that express both PrP constructs were left untreated or were incubated with CsA.…”

“…Those observations indicate that the entry of PrP into the ER is a prerequisite for its secretion by exosomes upon exposure to CsA and that cytosolic PrP is secreted from cells by different vesicles, potentially exosomes, whose secretion is independent of CsA. Next, we sought to explore whether the GPI tail of PrP, which anchors it to lipid rafts and is required for the deposition of CsA-induced misfolded PrP in aggresomes (17), is needed for the secretion of PrP by exosomes. N2a cells were transfected with a mutated PrP that carries the natural PrP ER-localization signal and a stop codon at residue 228 and thus, enters the ER but bears no GPI anchor.…”

“…Previously, we discovered that those proline substitutions prevent the interaction of PrP with the endoplasmic reticulum (ER)-resident chaperone cyclophilin B, thereby causing PrP misfolding and aggregation (4,16). Those misfolded PrP molecules are exported from the ER (17) and deposited in cytosolic sites called aggresomes (16), which attract molecular chaperones and proteasomes and serve as dynamic quality-control compartments. Similarly, cyclophilin inhibition by the fungal metabolite cyclosporin A (CsA) induces PrP misfolding, aggregation and deposition in aggresomes (16) but has no detectable effect on proteasome activity (4).…”