Dissection of the mechanism for the stringent factor RelA

During conditions of nutrient deprivation, ribosomes are blocked by uncharged tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but independently of ribosomal protein L11, while (p)ppGpp synthesis strictly requires uncharged tRNA at the A site and the presence of L11. A model is proposed whereby RelA hops between blocked ribosomes, providing an explanation for how low intracellular concentrations of RelA (1/200 ribosomes) can synthesize (p)ppGpp at levels that accurately reflect the starved ribosome population.     

The effect of ICAD-S on the formation and intracellular distribution of a nucleolytically active caspase-activated DNase

We show here that co-expression of murine CAD with either ICAD-L or ICAD-S in Escherichia coli as well as mammalian cells leads to a functional DFF complex, which after caspase-3 activation releases a nucleolytically active DNase. The chaperone activity of ICAD-S is between one and two orders of magnitude less effective than that of ICAD-L, as deduced from cleavage experiments with different activated recombinant DFF complexes produced in E.coli. With nucleolytically active EGFP fusion proteins of CAD it is demonstrated that co-expression of ICAD-S, which lacks the C-terminal domain of ICAD-L, including the NLS, leads to a homogeneous intracellular distribution of the DNase in transfected cells, whereas co-expression of human or murine ICAD-L variants lacking the NLS leads to exclusion of EGFP–CAD from the nuclei in ~50% of cells. These results attribute a particular importance of the NLS in the long isoform of the inhibitor of CAD for nuclear accumulation of the DFF complex in living cells. It is concluded that ICAD-L and ICAD-S in vivo might function as tissue-specific modulators in the regulation of apoptotic DNA degradation by controlling not only the enzymatic activity but also the amount of CAD available in the nuclei of mammalian cells.     

The Pco proteins are involved in periplasmic copper handling in Escherichia coli

The interactions between the plasmid-borne copper resistance determinant, pco, and the main copper export system in Escherichia coli have been investigated and no direct interaction has been found. The PcoE and PcoC proteins are periplasmic and PcoC binds one Cu ion per protein molecule. PcoA is also periplasmic and can substitute for the chromosomally encoded CueO protein. The pco determinant is proposed to exert its effect through periplasmic handling of excess copper ions and to increase the level of resistance to copper ions above that conferred by copA alone.     

Zinc-dependent interaction between dishevelled and the Drosophila Wnt antagonist naked cuticle

During Drosophila development, the naked cuticle (nkd) gene attenuates wingless/Wnt signaling through a negative feedback loop mechanism. Fly and vertebrate Nkd proteins contain a putative calcium-binding EF-hand motif, the EFX domain, that interacts with the basic/PDZ region of the Wnt signal transducer, dishevelled (Dsh). Here we show that Dsh binding by Drosophila Nkd in vitro is mediated by the EFX domain as well as an adjacent C-terminal sequence. In vivo data suggest that both of these regions contribute to the ability of Nkd to antagonize Wnt signaling. Mutations in the Nkd EF-hand designed to eliminate potential ion binding affected Nkd-Dsh interactions in the yeast two-hybrid assay but not in the glutathione S-transferase pull-down assay. Addition of the chelating agent EDTA abolished the in vitro Nkd-Dsh interaction. Surprisingly zinc, but not calcium, was able to restore Nkd-Dsh binding, suggesting a zinc-mediated interaction. Calcium 45- and zinc 65-blotting experiments show that Nkd is a zinc-binding metalloprotein. The results further clarify how Nkd may antagonize Wnt signaling via interaction with Dsh, and identify a novel zinc-binding domain in Drosophila Nkd that collaborates with the conserved EFX domain to bind Dsh.     

Isolation and characterization of the Spindly homologue from tomato

The SPINDLY (SPY) gene is a crucial component of the gibberellin signal transduction pathway in Arabidopsis thaliana (L.) Heynh. In this study, the cloning of the SPINDLY-orthologous gene (LeSpy) from tomato (Lycopersicon esculentum Mill.) and its characterization are reported. SPY and LeSpy show high sequence similarity along their entire lengths, which is reflected in the conservation of exon-intron structure and of all sequence motives previously identified. To analyse the relationship between the Arabidopsis spindly and the tomato procera mutant, which show similar phenotypes, the LeSpy gene was characterized in wild-type and procera tomato plants. These analyses as well as mapping of LeSpy revealed that LeSpy and Procera are different genes.     

Novel functional role of CA repeats and hnRNP L in RNA stability

CA dinucleotide repeat sequences are very common in the human genome. We have recently demonstrated that the polymorphic CA repeats in intron 13 of the human endothelial nitric oxide synthase (eNOS) gene function as an unusual, length-dependent splicing enhancer. The CA repeat enhancer requires for its activity specific binding of hnRNP L. Here we show that in the absence of bound hnRNP L, the pre-mRNA is cleaved directly upstream of the CA repeats. The addition of recombinant hnRNP L restores RNA stability. CA repeats are both necessary and sufficient for this specific cleavage in the 5' adjacent RNA sequence. We conclude that-in addition to its role as a splicing activator-hnRNP L can act in vitro as a sequence-specific RNA protection factor. Based on the wide abundance of CA repetitive sequences in the human genome, this may represent a novel, generally important role of this abundant hnRNP protein.     

No superoxide dismutase activity of cellular prion protein in vivo

Prion diseases are characterized by the deposition of PrP(Sc), an abnormal form of the cellular prion protein PrP(C), which is encoded by the Prnp gene. PrP(C) is highly expressed on neurons and its function is unknown. Recombinant PrP(C) was claimed to possess superoxide dismutase (SOD) activity, and it was hypothesized that abrogation of this function may contribute to neurodegeneration in prion diseases. We tested this hypothesis in vivo by studying copper/zinc and manganese SOD activity in genetically defined crosses of mice lacking the Sod1 gene with mice lacking PrP(C), and with hemizygous or homozygous tga20 transgenic mice overexpressing various levels of PrP(C). We failed to detect any influence of the Prnp genotype and gene dosage on SOD1 or SOD2 activity in heart, spleen, brain, and synaptosome-enriched brain fractions. Control experiments included crosses of mice lacking or overexpressing PrPc with mice overexpressing human Cu2+/Zn2+-superoxide dismutase, and confirmed that SOD enzymatic activity correlated exclusively with the gene dosage of bona fide human or murine SOD. We conclude that PrP(C) in vivo does not discernibly contribute to total SOD activity and does not possess an intrinsic dismutase activity.     

Phosphorylation of the MAPKKK regulator Ste50p in Saccharomyces cerevisiae: a casein kinase I phosphorylation site is required for proper mating function

The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase. Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals. Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself. Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo. Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I. Replacement of T42 with alanine (T42A) compromises Ste50p function. This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain. Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function. These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response. However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.