Changes in mitochondrial morphology that occur during cell cycle, differentiation, and death are tightly regulated by the balance between fusion and fission processes. Excessive fragmentation can be caused by inhibition of the fusion machinery and is a common consequence of dysfunction of the organelle. Here, we show a role for calcineurin-dependent translocation of the profission dynamin related protein 1 (Drp1) to mitochondria in dysfunction-induced fragmentation. When mitochondrial depolarization is associated with sustained cytosolic Ca 2؉ rise, it activates the cytosolic phosphatase calcineurin that normally interacts with Drp1. Calcineurindependent dephosphorylation of Drp1, and in particular of its conserved serine 637, regulates its translocation to mitochondria as substantiated by site directed mutagenesis. Thus, fragmentation of depolarized mitochondria depends on a loop involving sustained Ca 2؉ rise, activation of calcineurin, and dephosphorylation of Drp1 and its translocation to the organelle.fission ͉ phosphatase ͉ subcellular localization ͉ calcium ͉ cyclosporine A

The mammalian prion protein (PrPC) is a cell surface protein consisting of a flexibly disordered N-terminal segment (residues 23−120) and a structured C-terminal domain (residues 121−231). PrPC is supposed to bind Cu2+ in vivo, and several studies have recently focused on the ability of this protein to bind divalent cations. In a previous continuous wave electron paramagnetic resonance (CW EPR) study, we showed that Cu(II) binds both to the N- and C-terminal parts of PrPC. Here we present a pulse EPR and electron nuclear double resonance (ENDOR) study of the three different Cu(II) binding sites observed in the structured, C-terminal part of the murine prion protein, mPrP(121−231). It was found that the three complexes are distinguished by a different number of nitrogen atoms directly involved in the Cu(II) ligation. For one of the Cu(II) binding sites that is observed at low pH (3−6), no directly coupled nitrogens could be observed. For a second type of Cu(II) complex, observed at pH 3−8, Davies-ENDOR and hyperfine sublevel correlation (HYSCORE) spectroscopy revealed that histidine is one of the binding ligands. Furthermore, the presence of a nonexchangeable proton close to a copper ion could be demonstrated in a sample containing mainly the second Cu(II) complex. For the third mode of Cu(II) complexation, which can be detected at pH 7−8, Davies-ENDOR spectra indicate that more than one nitrogen atom is directly bound to the copper ion. The observed EPR parameters suggest the involvement of backbone nitrogens in this copper(II) complex.

Transmissible spongiform encephalopathies in mammals are believed to be caused by scrapie form of prion protein (PrP(Sc)), an abnormal, oligomeric isoform of the monomeric cellular prion protein (PrP(C)). One of the proposed functions of PrP(C) in vivo is a Cu(II) binding activity. Previous studies revealed that Cu(2+) binds to the unstructured N-terminal PrP(C) segment (residues 23-120) through conserved histidine residues. Here we analyzed the Cu(II) binding properties of full-length murine PrP(C) (mPrP), of its isolated C-terminal domain mPrP(121-231) and of the N-terminal fragment mPrP(58-91) in the range of pH 3-8 with electron paramagnetic resonance spectroscopy. We find that the C-terminal domain, both in its isolated form and in the context of the full-length protein, is capable of interacting with Cu(2+). Three Cu(II) coordination types are observed for the C-terminal domain. The N-terminal segment mPrP(58-91) binds Cu(2+) only at pH values above 5.0, whereas both mPrP(121-231) and mPrP(23-231) already show identical Cu(II) coordination in the pH range 3-5. As the Cu(2+)-binding N-terminal segment 58-91 is not required for prion propagation, our results open the possibility that Cu(2+) ions bound to the C-terminal domain are involved in the replication of prions, and provide the basis for further analytical studies on the specificity of Cu(II) binding by PrP.

No abstract