The RanBP2 zinc finger domains of chloroplast RNA editing factor OZ1 are required for protein–protein interactions and conversion of C to U

In the chloroplast, organelle zinc finger 1 (OZ1) is a RanBP2-type zinc finger (Znf) protein required for many RNA editing events, a process by which specific cytosines are enzymatically converted to uracils as a correction mechanism for missense mutations in the organelle genomes. RNA editing is carried out by a large multi-protein complex called the ‘editosome’ that contains members of the pentatricopeptide repeat (PPR) protein family, the RNA editing factor interacting protein (also known as MORF) family and the organelle RNA-recognition motif (ORRM) family, in addition to OZ1. OZ1 is an 82-kDa protein with distinct domains, including a pair of Znf domains and a unique C-terminal region. To elucidate the functions of these domains, we have generated truncations of OZ1 for use in protein–protein interaction assays that identified the C-terminal region of OZ1, as well as the Znf domains as the primary interactors with PPR proteins, which are factors required for site-specificity and enzymatic editing. Expression of these OZ1 truncations in vivo showed that the Znf domains were required to restore chloroplast RNA editing in oz1 knockout plants. Mutation of key structural residues in the Znf domains showed that they are necessary for editing and required for interaction with ORRM1, a general editing factor with an RNA-binding domain. These functional characterizations of the Znfs and novel C-terminal domain contribute to our understanding of the model for the chloroplast plant editosome.     

The effect of RNA interference Down-regulation of RNA editing 3'-terminal uridylyl transferase (TUTase) 1 on mitochondrial de novo protein synthesis and stability of respiratory complexes in Trypanosoma brucei

Inhibition of RNA editing by down-regulation of expression of the mitochondrial RNA editing TUTase 1 by RNA interference had profound effects on kinetoplast biogenesis in Trypanosoma brucei procyclic cells. De novo synthesis of the apocytochrome b and cytochrome oxidase subunit I proteins was no longer detectable after 3 days of RNAi. The effect on protein synthesis correlated with a decline in the levels of the assembled mitochondrial respiratory complexes III and IV, and also cyanide-sensitive oxygen uptake. The steady-state levels of nuclear-encoded subunits of complexes III and IV were also significantly decreased. Because the levels of the corresponding mRNAs were not affected, the observed effect was likely due to an increased turnover of these imported mitochondrial proteins. This induced protein degradation was selective for components of complexes III and IV, because little effect was observed on components of the F(1).F(0)-ATPase complex and on several other mitochondrial proteins.     

The alpha regulatory subunit of the mitochondrial F1-ATPase complex is a heat-shock protein. Identification of two highly conserved amino acid sequences among the alpha-subunits and molecular chaperones

The recent identification of the alpha-subunit of mitochondrial F1-ATPase complex in rat liver peroxisomes suggests another functional role for this protein in both organelles in addition to its involvement in mitochondrial oxidative phosphorylation. We report here that a very rapid response (15 min) in the induction of the alpha-regulatory subunit of the mitochondrial F1-ATPase complex is observed in 37 degrees C heat-shocked larvae of Drosophila hydei. Under the same heat-shock treatment, a similar-fold induction for the heat-shock protein hsp-70 was less rapid (45 min). Although the amino acid sequence identities between the "chaperonine" and the alpha-subunit protein families are very low (less than 20%), two amino acid sequences, of 12 and 13 residues each, are found in the alpha-subunits of the F1-ATPase complex from various eukaryotes which show a highly conserved identity (over 50%) with amino acid sequences found in molecular chaperones. We suggest that the nuclear coded alpha-subunit belongs to the family of stress proteins hsp-60 and thus, that it could perform similar functional role(s) to those recently described for mitochondrial hsp-60 (Cheng, M. Y., Hartl, F. U., Martin, J., Pollock, R. A., Kalousek, F., Neupert, W., Hallberg, E. M., Hallberg, R. L., and Horwich, A. L. (1989) Nature 337, 620-625 and Ostermann, J., Horwich, A. L., Neupert, W., and Ultrich-Hartl, F. (1989) Nature 341, 125-130) in both the mitochondria and the peroxisomes. Furthermore, we suggest that the two conserved elements among the chaperonines and the alpha-subunits could putatively be involved in the chaperonine function of these proteins.     

Macrophage colony-stimulating factor expression in retrovirally transduced cells is dependent upon both the adherence status of the target cells and its 5' flanking untranslated region

Numerous cell types retrovirally transduced with macrophage colony-stimulating factor (M-CSF) using LXSN-based vectors showed a variable expression of the transgene. Expression of M-CSF correlated with the cells' adherent status. Transduced adherent cells produced the M-CSF, whereas the non-adherent cells synthesized little M-CSF. Studies showed that the 5'-UTR of the M-CSF gene regulated transgenic M-CSF gene expression. Ligation of this 5'-UTR to the enhanced green fluorescent protein gene (EGFP) caused the expression of EGFP to show the same dichotomy as previously seen with the M-CSF. Transgenic M-CSF was expressed within non-adherent cells when the 5'-UTR was removed from the LXSN vector. Quantitative real-time polymerase chain reaction analysis confirmed that lesser production of M-CSF mRNA occurred within the non-adherent cells than in the adherent cells. This difference was eliminated when the 5'-UTR was removed from the retroviral vector. Our work suggests that this 5'-UTR of the M-CSF gene could be an important way to get transgenic expression within adherent cells, but not in non-adherent cells.     

The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. A fluorescence study.

The topography of membrane-bound blood coagulation factor IXa (fIXa) and the nature of its interaction with its cofactor, factor VIIIa (fVIIIa), were examined using fluorescent derivatives of fIXa. A fluorescein dye was covalently attached to the active-site histidine of fIXa via a D-Phe-Pro-Arg tripeptide tether to form Fl-A-FPR-fIXa; similarly, a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached via Glu-Gly-Arg to form DEGR-fIXa. When either Fl-A-FPR-fIXa or DEGR-fIXa was titrated with phosphatidylcholine-phosphatidylserine vesicles containing octadecylrhodamine in the presence of Ca2+, fluorescence energy transfer was observed. Assuming a random orientation of dyes, the distance of closest approach between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the membrane surface was found to be 89 +/- 3 A for Fl-A-FPR-fIXa and 73 +/- 4 A for DEGR-fIXa. Although the exact distance remains uncertain, it is clear that the active site of fIXa is positioned more than 70 A above the surface, and hence that the elongated fIXa molecule projects approximately perpendicularly from the surface when bound to the membrane. The binding of fVIIIa to membrane-bound Fl-A-FPR-fIXa or DEGR-fIXa did not alter the location of the active site relative to the membrane surface, but did alter both the emission intensity and anisotropy of the fluorescein and dansyl probes and hence their environments. Cofactor stimulation of fIXa activity therefore appears to be mediated, at least in part, by a conformational change in the active site that occurs when fVIIIa binds to the enzyme on the phospholipid surface.     

The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an axonemal protein widely conserved among organisms with motile cilia and flagella

The gene product of EFHC1 recently implicated in juvenile myoclonic epilepsy (JME) was found to be a homolog of Chlamydomonas axonemal protein Rib72, whose homologs are present in a wide variety of organisms that have motile cilia and flagella. Western blot analyses and immunofluorescence localization of the mouse ortholog mRib72-1/Efhc1 indicated that it is indeed abundantly present in sperm flagella and tracheal cilia but only in a small amount in the brain. It is not present in immotile primary cilia. These observations raise the possibility that malfunction of motile cilia is involved in the development of JME.     

The AsaP1 peptidase of Aeromonas salmonicida subsp. achromogenes is a highly conserved deuterolysin metalloprotease (family M35) and a major virulence factor

Infections by the bacterium Aeromonas salmonicida subsp. achromogenes cause significant disease in a number of fish species. In this study, we showed that AsaP1, a toxic 19-kDa metallopeptidase produced by A. salmonicida subsp. achromogenes, belongs to the group of extracellular peptidases (Aeromonas type) (MEROPS ID M35.003) of the deuterolysin family of zinc-dependent aspzincin endopeptidases. The structural gene of AsaP1 was sequenced and found to be highly conserved among gram-negative bacteria. An isogenic ΔasaP1 A. salmonicida subsp. achromogenes strain was constructed, and its ability to infect fish was compared with that of the wild-type (wt) strain. The ΔasaP1 strain was found to infect Arctic charr, Atlantic salmon, and Atlantic cod, but its virulence was decreased relative to that of the wt strain. The 50% lethal dose of the AsaP1 mutant was 10-fold higher in charr and 5-fold higher in salmon than that of the wt strain. The pathology induced by the AsaP1-deficient strain was also different from that of the wt strain. Furthermore, the mutant established significant bacterial colonization in all observed organs without any signs of a host response in the infected tissue. AsaP1 is therefore the first member of the M35 family that has been shown to be a bacterial virulence factor.