High-throughput identification of protein localization dependency networks

Bacterial cells are highly organized with many protein complexes and DNA loci dynamically positioned to distinct subcellular sites over the course of a cell cycle. Such dynamic protein localization is essential for polar organelle development, establishment of asymmetry, and chromosome replication during the Caulobacter crescentus cell cycle. We used a fluorescence microscopy screen optimized for high-throughput to find strains with anomalous temporal or spatial protein localization patterns in transposon-generated mutant libraries. Automated image acquisition and analysis allowed us to identify genes that affect the localization of two polar cell cycle histidine kinases, PleC and DivJ, and the pole-specific pili protein CpaE, each tagged with a different fluorescent marker in a single strain. Four metrics characterizing the observed localization patterns of each of the three labeled proteins were extracted for hundreds of cell images from each of 854 mapped mutant strains. Using cluster analysis of the resulting set of 12-element vectors for each of these strains, we identified 52 strains with mutations that affected the localization pattern of the three tagged proteins. This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. These studies provide insights into factors affecting the PleC/DivJ localization network and into regulatory links between the localization of the pili assembly protein CpaE and the kinase localization pathway. Our high-throughput screening methodology can be adapted readily to any sequenced bacterial species, opening the potential for databases of localization regulatory networks across species, and investigation of localization network phylogenies.     

Identification of a localization factor for the polar positioning of bacterial structural and regulatory proteins

Polar pili biogenesis in Caulobacter involves the asymmetric localization of the CpaE and CpaC components of the pili-specific secretion apparatus to one pole of the predivisional cell followed by the biosynthesis of the pili filaments in the daughter swarmer cell. The histidine kinase signaling protein, PleC, that controls the temporal accumulation of the PilA pilin subunit is asymmetrically localized to the pole at which pili are assembled. Here we identify a protein, PodJ, that provides the positional information for the polar localization of both PleC and CpaE. The PodJ protein was found to exist in two forms, a truncated 90-kDa and a full-length 110-kDa form, each controlling a different aspect of polar development and each localizing to the cell poles at a specific time in the cell cycle. When active PleC is delocalized in a DeltapodJ mutant, the accumulation of PilA, the downstream target of PleC signaling, is impaired, providing evidence that the polar localization of this histidine kinase stimulates the response signaled by a two-component system.     

CheA protein, a central regulator of bacterial chemotaxis, belongs to a family of proteins that control gene expression in response to changing environmental conditions.

During bacterial chemotaxis, the binding of stimulatory ligands to chemoreceptors at the cell periphery leads to a response at the flagellar motor. Three proteins appear to be required for receptor-mediated control of swimming behavior, the products of the cheA, cheW, and cheY genes. Here we present the complete nucleotide sequence of the Salmonella typhimurium cheA gene together with the purification and characterization of its protein product. The protein is a 73,000 Mr cytoplasmic constituent. Amino acid-sequence comparisons indicate that it belongs to a family of bacterial regulatory proteins including the products of the cpxA, dctB, envZ, ntrB, phoR, phoM, and virA genes. Each member of this family has a conserved domain of approximately equal to 200 residues within its C terminus. We have previously shown that another chemotaxis protein, CheY, represents a domain of protein structure that has been conserved within a second large family of bacterial regulatory proteins. Each protein of the CheA family seems to function as a regulator of a different CheY homologue. Although each pair of proteins appears to produce a specialized response to a distinct type of stimulus, the relationships in primary structure suggest that a similar molecular mechanism may be involved.     

Polar auxin transport in the wood-forming tissues of hybrid aspen is under simultaneous control of developmental and environmental signals

Recent research has highlighted the importance of auxin concentration gradients during plant development. Establishment of these gradients is believed to involve polar auxin transport through specialized carrier proteins. We have used an experimental system, the wood-forming tissue of hybrid aspen, which allows tissue-specific expression analysis of auxin carrier genes and quantification of endogenous concentrations of the hormone. As part of this study, we isolated the putative polar auxin transport genes, PttLAX1-PttLAX3 and PttPIN1-PttPIN3, belonging to the AUX1-like family of influx and PIN1-like efflux carriers, respectively. Analysis of PttLAX and PttPIN expression suggests that specific positions in a concentration gradient of the hormone are associated with different stages of vascular cambium development and expression of specific members of the auxin transport gene families. We were also able demonstrate positive feedback of auxin on polar auxin transport genes. Entry into dormancy at the end of a growing season leads to a loss of auxin transport capacity, paralleled by reduced expression of PttLAX and PttPIN genes. Furthermore, data from field experiments show that production of the molecular components of the auxin transport machinery is governed by environmental controls. Our findings collectively demonstrate that trees have developed mechanisms to modulate auxin transport in the vascular meristem in response to developmental and environmental cues.     

Ribosomal protein L11 mutation in zebrafish leads to haematopoietic and metabolic defects

Mutations in ribosomal proteins are associated with a congenital syndrome, Diamond-Blackfan anaemia (DBA), manifested by red blood cell aplasia, developmental abnormalities and increased risk of malignancy. Recent studies suggest the involvement of p53 activation in DBA. However, which pathways are involved and how they contribute to the DBA phenotype remains unknown. Here we show that a zebrafish mutant for the rpl11 gene had defects both in the development of haematopoietic stem cells (HSCs) and maintenance of erythroid cells. The molecular signature of the mutant included upregulation of p53 target genes and global changes in metabolism. The changes in several pathways may affect haematopoiesis including upregulation of pro-apoptotic and cell cycle arrest genes, suppression of glycolysis, downregulation of biosynthesis and dysregulation of cytoskeleton. Each of these pathways has been individually implicated in haematological diseases. Inhibition of p53 partially rescued haematopoiesis in the mutant. Altogether, we propose that the unique phenotype of DBA is a sum of several abnormally regulated molecular pathways, mediated by the p53 protein family and p53-independent, which have synergistic impact on haematological and other cellular pathways affected in DBA. Our results provide new insights into the pathogenesis of DBA and point to the potential avenues for therapeutic intervention.     

Visualization of the movement of single histidine kinase molecules in live Caulobacter cells

The bacterium Caulobacter crescentus divides asymmetrically as part of its normal life cycle. This asymmetry is regulated in part by the membrane-bound histidine kinase PleC, which localizes to one pole of the cell at specific times in the cell cycle. Here, we track single copies of PleC labeled with enhanced yellow fluorescent protein (EYFP) in the membrane of live Caulobacter cells over a time scale of seconds. In addition to the expected molecules immobilized at one cell pole, we observed molecules moving throughout the cell membrane. By tracking the positions of these molecules for several seconds, we determined a diffusion coefficient (D) of 12 +/- 2 x 10(-3) microm(2)/s for the mobile copies of PleC not bound at the cell pole. This D value is maintained across all cell cycle stages. We observe a reduced D at poles containing localized PleC-EYFP; otherwise D is independent of the position of the diffusing molecule within the bacterium. We did not detect any directional bias in the motion of the PleC-EYFP molecules, implying that the molecules are not being actively transported.     

Building a human kinase gene repository: bioinformatics, molecular cloning, and functional validation

Kinases catalyze the phosphorylation of proteins, lipids, sugars, nucleosides, and other important cellular metabolites and play key regulatory roles in all aspects of eukaryotic cell physiology. Here, we describe the mining of public databases to collect the sequence information of all identified human kinase genes and the cloning of the corresponding ORFs. We identified 663 genes, 511 encoding protein kinases, and 152 encoding nonprotein kinases. We describe the successful cloning and sequence verification of 270 of these genes. Subcloning of this gene set in mammalian expression vectors and their use in high-throughput cell-based screens allowed the validation of the clones at the level of expression and the identification of previously uncharacterized modulators of the survivin promoter. Moreover, expressions of the kinase genes in bacteria, followed by autophosphorylation assays, identified 21 protein kinases that showed autocatalytic activity. The work described here will facilitate the functional assaying of this important gene family in phenotypic screens and their use in biochemical and structural studies.     

In vivo analysis of an essential myosin light chain mutation linked to familial hypertrophic cardiomyopathy

Mutations in cardiac motor protein genes are associated with familial hypertrophic cardiomyopathy. Mutations in both the regulatory (Glu22Lys) and essential light chains (Met149Val) result in an unusual pattern of hypertrophy, leading to obstruction of the midventricular cavity. When a human genomic fragment containing the Met149Val essential myosin light chain was used to generate transgenic mice, the phenotype was recapitulated. To unambiguously establish a causal relationship for the regulatory and essential light chain mutations in hypertrophic cardiomyopathy, we generated mice that expressed either the wild-type or mutated forms, using cDNA clones encompassing only the coding regions of the gene loci. Expression of the proteins did not lead to a hypertrophic response, even in senescent animals. Changes did occur at the myofilament and cellular levels, with the myofibrils showing increased Ca(2+) sensitivity and significant deficits in relaxation in a transgene dose-dependent manner. Clearly, mice do not always recapitulate important aspects of human hypertrophy. However, because of the discordance of these data with data obtained in transgenic mice containing the human genomic fragment, we believe that the concept that these point mutations by themselves can cause hypertrophic cardiomyopathy should be revisited.     

Why chloroplasts and mitochondria retain their own genomes and genetic systems: Colocation for redox regulation of gene expression

Chloroplasts and mitochondria are subcellular bioenergetic organelles with their own genomes and genetic systems. DNA replication and transmission to daughter organelles produces cytoplasmic inheritance of characters associated with primary events in photosynthesis and respiration. The prokaryotic ancestors of chloroplasts and mitochondria were endosymbionts whose genes became copied to the genomes of their cellular hosts. These copies gave rise to nuclear chromosomal genes that encode cytosolic proteins and precursor proteins that are synthesized in the cytosol for import into the organelle into which the endosymbiont evolved. What accounts for the retention of genes for the complete synthesis within chloroplasts and mitochondria of a tiny minority of their protein subunits? One hypothesis is that expression of genes for protein subunits of energy-transducing enzymes must respond to physical environmental change by means of a direct and unconditional regulatory control—control exerted by change in the redox state of the corresponding gene product. This hypothesis proposes that, to preserve function, an entire redox regulatory system has to be retained within its original membrane-bound compartment. Colocation of gene and gene product for redox regulation of gene expression (CoRR) is a hypothesis in agreement with the results of a variety of experiments designed to test it and which seem to have no other satisfactory explanation. Here, I review evidence relating to CoRR and discuss its development, conclusions, and implications. This overview also identifies predictions concerning the results of experiments that may yet prove the hypothesis to be incorrect.     

Cloning of Nrf1, an NF-E2-related transcription factor, by genetic selection in yeast.

The human homolog of mouse NF-E2 was isolated from the K562 cell line and found to encode a member of the basic leucine-zipper family of DNA-binding regulatory proteins. The deduced amino acid sequence of the mouse and human proteins exhibited near identity. Comparison to the related protein, Nrf1, revealed significant homologies at isolated regions, particularly within the basic domain, suggesting that NF-E2 and Nrf1 are members of a distinct subfamily of basic leucine-zipper proteins that share similar DNA-binding properties. High levels of human NF-E2 mRNA were observed in human erythroleukemic cell lines examined. Extensive survey of human tissue samples found NF-E2 expression not limited to erythropoeitic organs. Expression in the colon and testis suggests that NF-E2 may participate in the regulation of genes other than globin.