Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite’s life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion.     

The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D

The Minute phenotype results from mutations at?>50 loci scattered throughout the genome of Drosophila. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. Here, we report a novel P-element induced Minute mutation, P{lacW}M(3)66D ¹ , that maps to region 66D on chromosome 3L. Flies heterozygous for P{lacW}M(3)66D ¹ have a strong Minute phenotype. Molecular characterisation of the chromosomal region revealed three previously undescribed Drosophila genes clustered within a 5-kb genomic fragment. Two of the genes have significant sequence homology to genes for the mammalian ribosomal proteins L14 and RD, respectively, and share a joint 240-bp promoter region harbouring the P-element insert. Quantitative Northern blot analyses showed the mutation to affect RPL14 mRNA levels only. Interestingly, the reduction in abundance of RPL14 mRNA is not constitutive, indicating that the promoter function abolished by the inserted P-element is utilised with different efficiencies in different developmental situations. Remobilisation of the P element produced wild-type flies with normal levels of RPL14 mRNA, demonstrating that the mutant phenotype is caused by the insertion. P{lacW}M(3)66D ¹ joins a growing list of Minute mutations associated with ribosomal protein-haploinsufficiency.     

Transposable element-induced mutations of the viviparous-1 gene in maize

The viviparous-1 (vp1) locus in maize is a developmental gene that controls diverse aspects of the maturation phase of seed development. Mutations of vp1 alter embryo sensitivity to the hormone abscisic acid and block formation of anthocyanin pigment. Molecular cloning of a Robertson Mutator-induced mutant allele, vp1-mum-1, by transposable element tagging has allowed analysis of several transposon-induced vp1 mutants. In the vp1-Mc mutation, the gene is disrupted by 4.0 kbp insertion, which results in expression of a 3′ truncated mRNA. Phenotypically, this allele is at least partially functional in causing embryo dormancy, but is ineffective in controlling anthocyanin expression. This result suggests that disruption of the C-terminal domain of the Vp1 protein specifically affects regulation of the anthocyanin pathway. A second Mutator- derived allele, vp1-mum2, exhibits an unusual form of somatic mutability in which endosperm cells revert from wild-type vp1 expression to a mutant condition. The vp1-mum2 allele contains a 1.5 kbp Insertion that has no detectable homology to known Mu elements. This element is retained In wild-type germinal revertants derived from vp1-mum2 An apparent DNA modification affecting cleavage at an internal Sstl restriction site in the element correlates with vp1-mum2 states that exhibit wild-type Vp1 expression. A model involving mitotic assortment of modified and unmodified DNA strands during development is proposed for vp1-mum2 somatic mutation.     

Metabolism of thyrotropin releasing factor in two clonal cell lines of nervous system origin

Immunoreactive thyrotropin releasing factor (TRF) was detected in homogenates of two clonal cell lines, BN1010-1 and BN1010-3, derived from a rat central nervous system tumor. TRF was present in logarithmically-growing cells; daily medium changes with slightly acid culture medium (pH 6.8) greatly increased the TRF content of these cells. In contrast, TRF could not be detected in stationary phase cells. TRF peptidases were <1% as active in homogenates of BN1010 cells as those in homogenates of guinea pig brain or hypothalamus. It is expected that these cells will provide an excellent model system for the study of various aspects of TRF metabolism.     

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.