Lipochitooligosaccharide-induced tobacco cells release a peptide as mediator of the glycolipid signal

Lipochitooligosaccharides (LCOs) are plant growth regulators that promote at subfemtomolar concentrations cell division in tobacco protoplasts. In response to LCO treatment, tobacco cells release a second growth factor that fully mediates the growth-promoting activities of the initial extracellular LCO stimulus. This diffusible growth factor was isolated from the protoplasts’ culture filtrate and shown to be a peptide. We report that the LCO-induced mitogen released by tobacco cells and a synthetic heptadecapeptide derived from region 2 of the tobacco homolog of the early nodulin gene ENOD40 are antigenically related and qualitatively indistinguishable in their ability to stimulate cell division.

Identification and characterization of a conserved family of protein serine/threonine phosphatases homologous to Drosophila retinal degeneration C (rdgC)

The Drosophila retinal degeneration C (rdgC) gene encodes an unusual protein serine/threonine phosphatase in that it contains at least two EF-hand motifs at its carboxy terminus. By a combination of large-scale sequencing of human retina cDNA clones and searches of expressed sequence tag and genomic DNA databases, we have identified two sequences in mammals [Protein Phosphatase with EF-hands-1 and 2 (PPEF-1 and PPEF-2)] and one in Caenorhabditis elegans (PPEF) that closely resemble rdgC. In the adult, PPEF-2 is expressed specifically in retinal rod photoreceptors and the pineal. In the retina, several isoforms of PPEF-2 are predicted to arise from differential splicing. The isoform that most closely resembles rdgC is localized to rod inner segments. Together with the recently described localization of PPEF-1 transcripts to primary somatosensory neurons and inner ear cells in the developing mouse, these data suggest that the PPEF family of protein serine/threonine phosphatases plays a specific and conserved role in diverse sensory neurons.

Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events

Yeast two-hybrid and genetic interaction screens indicate that Bir1p, a yeast protein containing phylogenetically conserved antiapoptotic repeat domains called baculovirus inhibitor of apoptosis repeats (BIRs), is involved in chromosome segregation events. In the two-hybrid screen, Bir1p specifically interacts with Ndc10p, an essential component of the yeast kinetochore. Although Bir1p carries two BIR motifs in the N-terminal region, the C-terminal third of the protein is sufficient to provide strong interaction with Ndc10p and moderate interaction with Skp1p, another essential component of the yeast kinetochore. In addition, deletion of BIR1 is synthetically lethal with deletion of CBF1 or CTF19, genes specifying two other components of the yeast kinetochore. Yeast cells deleted of BIR1 have a chromosome-loss phenotype, which can be completely rescued by elevating NDC10 dosage. Furthermore, overexpression of either full-length or the C-terminal region of Bir1p can efficiently suppress the chromosome-loss phenotype of both bir1Δ null and skp1-4 mutants. Our data suggest that Bir1p participates in chromosome segregation events, either directly or via interaction with kinetochore proteins, and these effects are apparently not mediated by the BIR domains of Bir1p.

Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector λYES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel “APS reductase” enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of ≈1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.

Convergent pathways for lipochitooligosaccharide and auxin signaling in tobacco cells

Lipochitooligosaccharides (LCOs) are a novel class of plant growth regulators that activate in tobacco protoplasts the expression of AXI1, a gene implicated in auxin signaling. Transient assays with a chimeric PAXI-GUS expression plasmid revealed that the N-octadecenoylated monosaccharide GlcN has all structural requirements for a biological active glycolipid, whereas the inactive N-acylated GalN epimer inhibits LCO action. Specific inhibition of LCO and auxin action shows that both signals are transduced within the tobacco cell via separate pathways that converge at or before AXI1 transcription. Cytokinin is suggested to be a common effector of LCO and auxin signaling. We also show that activation of AXI1 correlates with growth factor-induced cell division.

Overexpression of a Novel Rho Family GTPase, RacC, Induces Unusual Actin-based Structures and Positively Affects Phagocytosis in Dictyostelium discoideum

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin–rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif–containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis in Dictyostelium.

A potential role for the nuclear factor of activated T cells family of transcriptional regulatory proteins in adipogenesis

NFAT (nuclear factor of activated T cells) is a family of transcription factors implicated in the control of cytokine and early immune response gene expression. Recent studies have pointed to a role for NFAT proteins in gene regulation outside of the immune system. Herein we demonstrate that NFAT proteins are present in 3T3-L1 adipocytes and, upon fat cell differentiation, bind to and transactivate the promoter of the adipocyte-specific gene aP2. Further, fat cell differentiation is inhibited by cyclosporin A, a drug shown to prevent NFAT nuclear localization and hence function. Thus, these data suggest a role for NFAT transcription factors in the regulation of the aP2 gene and in the process of adipocyte differentiation.

SARPs: A family of secreted apoptosis-related proteins

Quiescent mouse embryonic C3H/10T½ cells are more resistant to different proapoptotic stimuli than are these cells in the exponential phase of growth. However, the exponentially growing 10T½ cells are resistant to inhibitors of RNA or protein synthesis, whereas quiescent cells die upon these treatments. Conditioned medium from quiescent 10T½ cells possesses anti-apoptotic activity, suggesting the presence of protein(s) that function as an inhibitor of the apoptotic program. Using differential display technique, we identified and cloned a cDNA designated sarp1 (secreted apoptosis-related protein) that is expressed in quiescent but not in exponentially growing 10T½ cells. Hybridization studies with sarp1 revealed two additional family members. Cloning and sequencing of sarp2 and sarp3 revealed 38% and 40% sequence identity to sarp1, respectively. Human breast adenocarcinoma MCF7 cells stably transfected with sarp1 or infected with SARP1-expressing adenovirus became more resistant, whereas cells transfected with sarp2 displayed increased sensitivity to different proapoptotic stimuli. Expression of sarp family members is tissue specific. sarp mRNAs encode secreted proteins that possess a cysteine-rich domain (CRD) homologous to the CRD of frizzled proteins but lack putative membrane-spanning segments. Expression of SARPs modifies the intracellular levels of β-catenin, suggesting that SARPs interfere with the Wnt–frizzled proteins signaling pathway.

The MetaFam Server: a comprehensive protein family resource

MetaFam is a comprehensive relational database of protein family information. This web-accessible resource integrates data from several primary sequence and secondary protein family databases. By pooling together the information from these disparate sources, MetaFam is able to provide the most complete protein family sets available. Users are able to explore the interrelationships among these primary and secondary databases using a powerful graphical visualization tool, MetaFamView. Additionally, users can identify corresponding sequence entries among the sequence databases, obtain a quick summary of corresponding families (and their sequence members) among the family databases, and even attempt to classify their own unassigned sequences. Hypertext links to the appropriate source databases are provided at every level of navigation. Global family database statistics and information are also provided. Public access to the data is available at http://metafam.ahc.umn.edu/.