Loss of parkin function is linked to autosomal recessive juvenile parkinsonism. Here we show that proteotoxic stress and short C-terminal truncations induce misfolding of parkin. As a consequence, wild-type parkin was depleted from a high molecular weight complex and inactivated by aggregation. Similarly, the pathogenic parkin mutant W453Stop, characterized by a Cterminal deletion of 13 amino acids, spontaneously adopted a misfolded conformation. Mutational analysis indicated that C-terminal truncations exceeding 3 amino acids abolished formation of detergent-soluble parkin. In the cytosol scattered aggregates of misfolded parkin contained the molecular chaperone Hsp70. Moreover, increased expression of chaperones prevented aggregation of wild-type parkin and promoted folding of the W453Stop mutant. Analyzing parkin folding in vitro indicated that parkin is aggregation-prone and that its folding is dependent on chaperones. Our study demonstrates that C-terminal truncations impede parkin folding and reveal a new mechanism for inactivation of parkin.Autosomal recessive juvenile parkinsonism (AR-JP), 1 the major cause of early onset parkinsonism, is characterized by mutations within the parkin gene. Parkin, a 465-amino acid protein, shows homology to ubiquitin at the N terminus and harbors a RING box near the C terminus, consisting of two RING finger motifs that flank a cysteine-rich domain (in-between RING fingers domain) (1, 2). Functional studies established that parkin acts as a ubiquitin-protein isopeptide ligase and that pathogenic mutations compromise this activity (3-6). As a consequence, substrates destined for proteasomal degradation via parkin might accumulate in parkin-deficient cells. Indeed, recent studies with cell culture models provide experimental evidence for such a scenario. It was shown that disease-related mutations in the parkin gene impair protein interactions of parkin with either a parkin substrate or another component of the ubiquitin ligase complex. Accumulation of Pael-R, one of the identified parkin substrates, causes endoplasmic reticulum (ER) stress, indicating that parkin has the potential to suppress unfolded protein stress-induced cell death (3, 4). Recent studies (7) revealed that parkin deficiency potentiates the accumulation of cyclin E and promotes apoptosis in neuronal cells exposed to excitotoxic stress. Interestingly, parkin is a significant component of Lewy bodies, the histopathologic hallmark of PD (5,8,9). Furthermore, it has been shown that parkin is protective against the toxic effects of proteasomal dysfunction and mutant ␣-synuclein (10), implying that the impact of parkin function and dysfunction might not be restricted to the entity of AR-JP.It still remains enigmatic why dopaminergic neurons in the substantia nigra are highly vulnerable in AR-JP, as parkin as well as its substrates identified so far are not selectively expressed in these cells. However, an inherent feature of dopaminergic neurons is an elevated level of reactive oxygen and nitrogen species due to...

The mammalian prion protein (PrP) is composed of an unstructured flexible N-terminal region and a C-terminal globular domain. We examined the import of PrP into the endoplasmic reticulum (ER) of neuronal cells and show that information present in the C-terminal globular domain is required for ER import of the N terminus. N-terminal fragments of PrP, devoid of structural domains located in the C terminus, remained in the cytosol with an uncleaved signal peptide and were rapidly degraded by the proteasome. Conversely, the separate C-terminal domain of PrP, comprising the highly ordered helix 2-loop-helix 3 motif, was entirely imported into the ER. As a consequence, two PrP mutants linked to inherited prion disease in humans, PrPW145Stop and PrP-Q160Stop, were partially retained in the cytosol. The cytosolic fraction was characterized by an uncleaved N-terminal signal peptide and was degraded by the proteasome. Our study identified a new regulatory element in the C-terminal globular domain of PrP necessary and sufficient to promote import of PrP into the ER.

Misfolding of the mammalian prion protein (PrP) is implicated in the pathogenesis of prion diseases. We analyzed wild type PrP in comparison with different PrP mutants and identified determinants of the in vivo folding pathway of PrP. The complete N terminus of PrP including the putative transmembrane domain and the first ␤-strand could be deleted without interfering with PrP maturation. Helix 1, however, turned out to be a major determinant of PrP folding. Disruption of helix 1 prevented attachment of the glycosylphosphatidylinositol (GPI) anchor and the formation of complex N-linked glycans; instead, a high mannose PrP glycoform was secreted into the cell culture supernatant. In the absence of a C-terminal membrane anchor, however, helix 1 induced the formation of unglycosylated and partially protease-resistant PrP aggregates. Moreover, we could show that the C-terminal GPI anchor signal sequence, independent of its role in GPI anchor attachment, mediates core glycosylation of nascent PrP. Interestingly, conversion of high mannose glycans to complex type glycans only occurred when PrP was membrane-anchored. Our study indicates a bipartite function of helix 1 in the maturation and aggregation of PrP and emphasizes a critical role of a membrane anchor in the formation of complex glycosylated PrP.

N-linked glycans with complex structure have a major role in the biological activity of a wide variety of cell surface and secreted glycoproteins. Here, we show that geldanamycin, an inhibitor of Hsp90, interferes with the formation of complex glycosylated mammalian prion protein (PrP C ). Similarly to inhibitors of a-mannosidases, geldanamycin stabilized a high mannose PrP C glycoform and prevented the subsequent processing into complex structures. Moreover, a PrP/Grp94 complex could be isolated from geldanamycin-treated cells, suggesting that Grp94 might play a role in the processing of PrP C in the endoplasmic reticulum. Inhibition of complex glycosylation did not interfere with the glycosylphosphatidylinositol (GPI) anchor attachment and cellular trafficking of high mannose PrP C to the outer leaflet of the plasma membrane. In scrapie-infected neuroblastoma cells, however, high mannose PrP C glycoforms were preferred substrates for the formation of PrP-scrapie (PrP Sc ). Our study reveals that complex glycosylation is dispensable for the cellular trafficking of PrP C , but modulates the formation of PrP Sc .

Aberrant folding of the mammalian prion protein (PrP) is linked to prion diseases in humans and animals. We show that during post-translational targeting of PrP to the endoplasmic reticulum (ER) the putative transmembrane domain induces misfolding of PrP in the cytosol and interferes with its import into the ER. Unglycosylated and misfolded PrP with an uncleaved N-terminal signal sequence associates with ER membranes, and, moreover, decreases cell viability. PrP expressed in the cytosol, lacking the N-terminal ER targeting sequence, also adopts a misfolded conformation; however, this has no adverse effect on cell growth. PrP processing, productive ER import, and cellular viability can be restored either by deleting the putative transmembrane domain or by using a N-terminal signal sequence specific for co-translational ER import. Our study reveals that the putative transmembrane domain features in the formation of misfolded PrP conformers and indicates that post-translational targeting of PrP to the ER can decrease cell viability.Prion diseases in humans and animals are characterized by the accumulation of PrP Sc , a partially protease-resistant isoform of the cellular prion protein PrP C . PrP Sc is generated through a conformational transformation of PrP C and represents the major component of infectious prions (reviewed in Refs. 1-4). PrP 1 is post-translationally modified by the attachment of two N-linked complex carbohydrate moieties (Asn 180 and Asn 196 ) (5-7) and a glycosylphosphatidylinositol (GPI) anchor at serine 231 (8) as well as by the formation of a disulfide bond between Cys 178 and Cys 213 . Studies with recombinant PrP purified from bacteria revealed that the formation of the disulfide bond is essential for the native folding of PrP (9).The co-and post-translational modifications of PrP C are initiated with the cleavage of the N-terminal signal peptide (aa 1-22) and the transfer of core glycans, whereas the nascent chain is still associated with the translocon. Shortly after the protein is fully translocated, the GPI anchor is attached to the acceptor amino acid close to the C terminus. The final maturation of PrP C includes the processing of the core glycans into complex-type glycans by a series of enzymatic reactions in the endoplasmic reticulum (ER) and Golgi compartment. Posttranslational modifications, like N-linked glycosylation and GPI anchor attachment, are often used as diagnostic markers to monitor efficient import into the ER. In the case of PrP, however, we and others have shown that PrP devoid of a GPI anchor remains mainly unglycosylated but is imported efficiently into the ER and transported through the secretory pathway (10 -14). It has been found that the only specific marker for ER import of PrP is a cleaved N-terminal signal sequence (10). 2 Misfolding of PrP C in the cytosol or in the ER can induce neurodegeneration in the absence of PrP Sc . Neurotoxic properties of cytosolic PrP aggregates were observed after proteasomal inhibition in cultured cells or after the forced expr...