The central pathogenic event of prion disease is the conformational conversion of a host protein, PrP C , into a pathogenic isoform, PrP Sc . We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrP

Ran/TC4 is an essential, nuclear GTPase implicated in the initiation of DNA replication, entry into and exit from mitosis, and in nuclear RNA and protein transport through the nuclear pore complex. This diversity of functions suggests that Ran interacts with a large number of downstream targets. Using an overlay assay, we detected a family of putative target proteins that associate with GTP-bound Ran. The sequence of only one such protein, HTF9a/RanBPI, is known. We have now cloned two additional Ran-binding proteins, allowing identification of a distinctive, highly conserved sequence motif of 150 residues. This motif represents a minimal Ran-binding The Ran/TC4 GTPase is a highly conserved nuclear protein expressed in all eukaryotic cells examined to date (1-3). Disruption of Ran function, by either the introduction into cells of dominant gain-of-function mutants or removal of a regulatory factor, results in a pleiotropic phenotype characterized by cell-cycle arrest (4-8), premature chromosome condensation (2, 8) or exit from mitosis (9), and the accumulation of incorrectly processed nuclear RNA (10-12). In Xenopus oocyte preparations a dominant loss-of-function mutant of Ran blocks nuclear growth and the entry into S phase and inhibits dephosphorylation of the cdc2 p34 mitotic protein kinase (5), preventing the initiation of mitosis. In vitro studies also indicate that Ran may be an essential cytosolic component for the transport of karyophilic proteins into the nucleus through the nuclear-pore complex (13,14 MgCl2 (15). Unincorporated nucleotide was removed by passage over a Pharmacia PD-10 column. Plaques were lifted after induction for 4 hr with isopropyl f3-D-thiogalactoside and renatured overnight in 20 mM Hepes, pH 7.5/25 mM potassium acetate/10 mM magnesium acetate/5 mM dithiothreitol/50 ,tM GTP/50 ,uM GDP/4% nonfat dried milk/0.25% Tween 20. This was replaced with a similar buffer containing 0.05% Tween-20 and [a-32P]GTP-Ran (106 cpm/ml), and filters were incubated at 4°C for 2 hr and then washed five times in 150 mM NaCl/20 mM Tris, pH 7.4/10 mM magnesium acetate. pBluescript SK plasmids were excised from tertiary plaques according to the manufacturer's instructions (Stratagene). As a final test for clones that express Ran-binding proteins, bacteria containing plasmids with putative positive inserts were incubated with isopropyl P3-D-thiogalactoside to induce protein expression, and then bacterial extracts were run on SDS/ PAGE and transferred to nitrocellulose. RanBPs were detected by a Ran overlay assay as described (15). Two independent clones were identified that expressed RanBPs of -50 kDa (clone AB1) and 90 kDa (clone AB2) (data not shown). Inserts were completely sequenced in both directions by using an ABI automated sequencer.Expression and Analysis of the AB1 RBD. The putative Ran-binding domain of clone AB1 (390 bp, residues PHFE ... CKFE) was amplified by PCR and subcloned into pGEX-2T. The resulting glutathione S-transferase (GST) fusion protein was expressed in Escherichia coli...

Previously, we have identified a class of retinal degeneration mutants in Drosophila in which the normally transient interaction between arrestin2 (Arr2) and rhodopsin is stabilized and the complexes are rapidly internalized into the cell body by receptor-mediated endocytosis. The accumulation of protein complexes in the cytoplasm eventually results in photoreceptor cell death. We now show that the endocytic adapter protein AP-2 is essential for rhodopsin endocytosis through an Arr2-AP-2β interaction, and mutations in Arr2 that disrupt its interaction with the β subunit of AP-2 prevent endocytosis-induced retinal degeneration. We further demonstrate that if the interaction between Arr2 and AP-2 is blocked, this also results in retinal degeneration in an otherwise wild-type background. This indicates that the Arr2-AP-2 interaction is necessary for the pathology observed in a number of Drosophila visual system mutants, and suggests that regular rhodopsin turnover in wild-type photoreceptor cells by Arr2-mediated endocytosis is essential for photoreceptor cell maintenance.

The structural conversion of a host protein, PrP C , into a protease-resistant isoform, PrPres, is the central event in the pathogenesis of infectious prion diseases. Purification of native PrP C molecules from hamster brain by either cation exchange or immobilized chelator chromatographic resins yielded preparations that supported efficient amplification of scrapie-induced PrPres in vitro. Using these purified preparations, we determined that in vitro PrPres amplification was inhibited by CuCl 2 and ZnCl 2 at IC 50 concentrations of 400 nM and 10 lM, respectively. In contrast, 100 lM MnCl 2 did not directly inhibit PrPres amplification or block Cu 2+ -mediated inhibition. Additionally, the inhibition of PrPres amplification by Cu 2+ ions could be reversed by addition of either neocuproine or imidazole. Cu 2+ inhibited PrPres amplification in both the presence and absence of stimulatory polyanion molecules. These biochemical findings support the hypothesis that Cu 2+ ions might regulate the pathogenesis of prion diseases in vivo.

Previously we have shown that a subset of visual transduction mutants in Drosophila melanogaster induce the formation of stable complexes between rhodopsin and arrestin. One such mutant is in a visual system-specific phospholipase C (PLC). The rhodopsin/arrestin complexes generated in PLC mutants induce massive retinal degeneration. Here we demonstrate that both arrestin and rhodopsin undergo light-dependent endocytosis in a PLC mutant background. Interestingly, the internalized rhodopsin is rapidly degraded, but the arrestin is fully stable. The data are discussed with respect to mechanisms of arrestin-mediated endocytosis and human retinal disease.