Probing phosphoinositide functions in signaling and membrane trafficking

The inositol phospholipids (PIs) comprise a family of eight species with different combinations of phosphate groups arranged around the inositol ring. PIs are among the most versatile signaling molecules known, with key roles in receptor-mediated signal transduction, actin remodeling and membrane trafficking. Recent studies have identified effector proteins and specific lipid-binding domains through which PIs signal. These lipid-binding domains can be used as probes to further our understanding of the spatial and temporal control of individual PI species. New layers of complexity revealed by the use of such probes include the occurrence of PIs at intracellular locations, the identification of phosphatidylinositol signaling hotspots and the presence of non-membrane pools of PIs in cell nuclei.     

Dof基因家族调节植物生长发育功能的研究进展

Dof（DNA binding with one finger）蛋白是一类植物特异性转录因子,通常含有200～400个氨基酸和2个主要结构域。该家族成员的N 末端为高度保守的单锌指Dof结构域,具有与DNA和蛋白质相互作用的双重功能,其C末端的氨基酸序列则较为多变,是Dof蛋白重要的特异转录调控结构域。研究表明,Dof蛋白作为转录激活物或阻遏物参与了多方面的植物生长发育过程。随着基因组测序技术的发展,已有大量的Dof基因从植物基因组数据库中鉴定出来。该文对近年来国内外有关Dof基因家族的结构特点、全基因组鉴定、蛋白互作以及生物学功能等方面的研究进展进行综述,以期为Dof转录因子的深入研究提供参考。     

Genomic isolation of genes encoding starch branching enzyme II (SBEII) in apple: toward characterization of evolutionary disparity in <Emphasis Type="Italic">Sbe</Emphasis>II genes between monocots and eudicots

Reproductive and storage tissues of many plants produce large amounts of serine proteinase inhibitors (PIs). The ornamental tobacco, Nicotiana alata, produces a series of 6 kDa chymotrypsin and trypsin inhibitors that accumulate to up to 30% of soluble protein in the stigma. These inhibitors are derived by proteolytic processing of two closely related multidomain precursor proteins. Using immunogold electron microscopy, we find that the stigmatic PIs accumulate in both the central vacuole and in the extracellular mucilage. Labelling with antibodies specific for the C-terminal vacuolar targeting peptide (VTS) of each precursor confirms earlier biochemical data showing that the VTS is removed during passage through the secretory pathway. We have isolated and characterised the extracellular population of PIs, which are largely identical to PIs isolated from whole stigmas and are functional inhibitors of serine proteases. Subcellular fractionation of immature stigmas reveals that a sub-population of the PI precursor protein is proteolytically processed within the endoplasmic reticulum. This proteolysis results in the removal of the vacuolar sorting information, causing secretion of this PI population. We propose a novel mechanism whereby a single gene product may be simultaneously trafficked to two separate compartments mediated by proteolysis early in the secretory pathway.     

Visualization of Phosphoinositides via the Development of the Transient Expression System of a Cyan Fluorescent Protein in the Red Alga Porphyra yezoensis

Phosphoinositides (PIs) play important roles in signal transduction pathways and the regulation of cytoskeleton and membrane functions in eukaryotes. Subcellular localization of individual PI derivative is successfully visualized in yeast, animal, and green plant cells using PI derivative-specific pleckstrin homology (PH) domains fused with a variety of fluorescent proteins; however, expression of fluorescent proteins has not yet been reported in any red algal cells. In the present study, we developed the system to visualize these PIs using human PH domains fused with a humanized cyan fluorescent protein (AmCFP) in the red alga Porphyra yezoensis. Plasma membrane localization of AmCFP fused with the PH domain from phospholipase Cδ1 and Akt1, but not Bruton’s tyrosine kinase, was observed in cell wall-free monospores, demonstrating the presence of phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4-bisphosphate in P. yezoensis cells. This is the first report of the successful expression of fluorescent protein and the monitoring of PI derivatives in red algal cells. Our system, based on transient expression of AmCFP, could be applicable for the analysis of subcellular localization of other proteins in P. yezoensis and other red algal cells.     

The CEA family: a system in transitional evolution?

The CEA family consists of two structurally and functionally distinct sub-groups; the group including CEA, NCA and CGM-6 which are cell surface-bound by phosphatidyl-inositol (PI) linkages, and the group of BGP splice variants which have trans-membrane and cytoplasmic domains. Although all CEA family members mediate intercellular adhesion in vitro, the PI-linked group show Ca++ and temperature independent adhesion whereas the BGP group show rapidly reversible Ca++ and temperature dependent adhesion. From the close alignment in cDNA nucleotide sequences between family members and between repeated domains in one family member, it is apparent that the CEA family is now rapidly evolving; in fact, analogs of only the trans-membrane BGP group have been found so far in the mouse. The addition of a new group of potent adhesion molecules to complex species at some time after the rodent radiation has strong evolutional implications, which are discussed.     

Comparative analysis of the Kekkon molecules,related members of the LIG superfamily

Leucine-rich repeats (LRRs) and immunoglobulin (Ig) domains represent two of the most abundant sequence elements in metazoan proteomes. Despite this prevalence, comparatively few molecules containing both LRR and Ig (LIG) modules exist, and fewer still have been functionally defined. One LIG whose function has been investigated is the Drosophila protein Kekkon1 (Kek1). In vivo studies have demonstrated a role for Kek1 in Epidermal Growth Factor Receptor (EGFR) signaling and have suggested a role in neuronal pathfinding. Kek1 is the founding member of the Kek family, a group of six Drosophila transmembrane proteins that contain seven LRRs and a single Ig in their extracellular domains. While this arrangement of domains predicts a possible role as cell adhesion molecules (CAMs), to date little is known about the function or evolutionary relationship of these additional Kek molecules. Here we report that orthologs of Kek1, Kek2, Kek5, and Kek6 exist in the mosquito, Anopheles gambiae, and the honeybee, Apis mellifera, indicating that this family has been conserved for ~300 million years of evolutionary time. Comparative sequence analyses reveal remarkable identity among these orthologs, primarily in their extracellular regions. In contrast, the intracellular regions are more divergent, exhibiting only small pockets of conservation. In addition, we provide support for the general notion that these molecules may share common functions as CAMs, by demonstrating that Kek family members can form homotypic and heterotypic complexes.Edited by D. TautzChristina M. MacLaren, Timothy A. Evans and Diego Alvarado contributed equally to this work     

The Sac domain-containing phosphoinositide phosphatases: structure, function, and disease

Phosphoinositides (PIs) have long been known to have an essential role in cell physiology. Their intracellular localization and concentration must be tightly regulated for their proper function. This spatial and temporal regulation is achieved by a large number of PI kinases and phosphatases that are present throughout eukaryotic species. One family of these enzymes contains a conserved PI phosphatase domain termed Sac. Although the Sac domain is homologous among different Sac domain-containing proteins, all appear to exhibit varied substrate specificity and subcellular localization. Dysfunctions in several members of this family are implicated in a range of human diseases such as cardiac hypertrophy, bipolar disorder, Down’s syndrome, Charcot-Marie-Tooth disease (CMT) and Amyotrophic Lateral Sclerosis (ALS). In plant, several Sac domain-containing proteins have been implicated in the stress response, chloroplast function and polarized secretion. In this review, we focus on recent findings in the family of Sac domain-containing PI phosphatases in yeast, mammal and plant, including the structural analysis into the mechanism of enzymatic activity, cellular functions, and their roles in disease pathophysiology.     

Synaptopodin family of natively unfolded, actin binding proteins: physical properties and potential biological functions

The synaptopodin family of proteins consists of at least 3 members: synaptopodin, the synaptopodin 2 proteins, and the synaptopodin 2-like proteins. Each family member has at least 3 isoforms that are produced by alternative splicing. Synaptopodin family members are basic proteins that are rich in proline and have little regular 2° or 3° structure at physiological temperature, pH and ionic strength. Like other natively unfolded proteins, synaptopodin family members have multiple binding partners including actin and other actin-binding proteins. Several members of the synaptopodin family have been shown to stimulate actin polymerization and to bundle actin filaments either on their own or in collaboration with other proteins. Synaptopodin 2 has been shown to accelerate nucleation of actin filament formation and to induce actin bundling. The actin polymerization activity is inhibited by Ca²⁺-calmodulin. Synaptopodin 2 proteins are localized in Z-bands of striated and heart muscle and dense bodies of smooth muscle cells. Depending on the developmental status and stress, at least one member of the synaptopodin family can occupy nuclei of some cells. Members of the synaptopodin 2 subfamily have been implicated in cancers.     

Oleosin-like proteins are not present on the surface of tobacco pollen

Pollen-stigma interactions on wet- and dry-type stigmas involve similar processes: the hydration of the pollen, followed by pollen tube growth and penetration of the stigma. Furthermore, in some species, identical molecules, namely lipids, are used to achieve this. In addition to lipids, oleosin-like proteins of the pollen coat of dry-type stigma plants have been shown to be involved in pollen-stigma interactions. However, little information is present about the proteins on the surface of pollen of wet-type stigma plants, in particular that of the Solanaceae. To analyze proteins from the surface of pollen of Nicotiana tabacum (tobacco), a solanaceous plant, we used an antiserum raised against Brassica pollen coat, a dry-type stigma plant of the Brassicaceae. In addition we used a molecular approach to identify tobacco homologues of oleosin-like genes. Our results show that no proteins similar to Brassica oleracea pollen coat proteins are present on the surface of tobacco pollen, and that oleosin-like genes are not expressed in tobacco anthers or stigmas.     

Genomic Structure and Chromosomal Mapping of the MousenovGene

Thenovgene encodes a cysteine-rich protein that is overexpressed in avian nephroblastomas. It is a member of the CCN family of proteins, all of which are involved in cell growth. Genomic and cDNA clones encompassing the mousenovgene have been isolated and characterized. The mousenovgene is highly conserved with the human and chicknovgenes at the level of nucleotide sequence and genomic organization. The exon structure reflects the modular organization of the NOV protein in a number of structural domains. These are highly conserved with other members of the CCN family, as is the distribution of 38 of its 40 cysteine residues. Thenovgene maps to chromosome 15, between D15 Mit 153 and D15 Mit 183, in a region of conserved synteny with human chromosome 8.     

A combination of the F-box motif and kelch repeats defines a large Arabidopsis family of F-box proteins

In the sequences released by the Arabidopsis Genome Initiative (AGI), we have discovered a new large gene family (48 genes as of July 2000). A detailed computational and biochemical analysis of the predicted gene products reveals a novel family of plant F-box proteins, where the amino (N)-terminal F-box motif is followed by four kelch repeats and a characteristic carboxy-terminal domain. F-box proteins are an expanding family of eukaryotic proteins, which have been shown in some cases to be critical for the controlled degradation of cellular regulatory proteins via the ubiquitin pathway. The F-box motif of the At5g48990 gene product, a member of the family, was shown to be functionally active by its ability to mediate the in vitro interaction between At5g48990 and ASK1 proteins. F-box proteins specifically recruit the targets to be ubiquitinated, mainly through protein-protein interaction modules such as WD-40 domains or leucine-rich repeats (LRRs). The kelch repeats of the family described here form a potential protein-protein interaction domain, as molecular modelling of the kelch repeats according to the galactose oxidase crystal structure (the only solved structure containing kelch repeats) predicts a -propeller. The identification of this family of F-box proteins greatly expands the field of plant F-box proteins and suggests that controlled degradation of cellular proteins via the ubiquitin pathway could play a critical role in multiple plant cellular processes.     

Two large Arabidopsis thaliana gene families are homologous to the Brassica gene superfamily that encodes pollen coat proteins and the male component of the self-incompatibility response

The male component of the self-incompatibility response in Brassica has recently been shown to be encoded by the S locus cysteine-rich gene (SCR). SCR is related, at the sequence level, to the pollen coat protein (PCP) gene family whose members encode small, cysteine-rich proteins located in the proteo-lipidic surface layer (tryphine) of Brassica pollen grains. Here we show that the Arabidopsis genome includes two large gene families with homology to SCR and to the PCP gene family, respectively. These genes are poorly predicted by gene-identification algorithms and, with few exceptions, have been missed in previous annotations. Based on sequence comparison and an analysis of the expression patterns of several members of each family, we discuss the possible functions of these genes. In particular, we consider the possibility that SCR-related genes in Arabidopsis may encode ligands for the S gene family of receptor-like kinases in this species.     

Localization of genes encoding two human one-domain members of the AAA family: PSMC5 (the thyroid hormone receptor-interacting protein, TRIP1) and PSMC3 (the Tat-binding protein, TBP1)

The eukaryotic genome contains a putative ATPase gene family that encodes proteins with one or two highly conserved domain(s) of approximately 230 amino acids. These proteins have diverse cellular functions and mutation in at least one member of the family has been associated with human disease, while mutations in other family members are known to cause cell cycle defects in yeast. Therefore it is of interest to map more family members and so we have localized PSMC5 (the thyroid hormone receptor-interacting protein, TRIP1) and PSMC3 (the Tat-binding protein, TBP1) to chromosomes 17q24– q25 and 11p12–p13, respectively. We also present the map position of a probable PSMC3 processed pseudogene locus on chromosome 9p. Received: 18 July 1996     

Evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase family of the enolase superfamily

Understanding how proteins evolve to provide both exquisite specificity and proficient activity is a fundamental problem in biology that has implications for protein function prediction and protein engineering. To study this problem, we analyzed the evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase (OSBS/NAAAR) family, part of the mechanistically diverse enolase superfamily. Although all characterized members of the family catalyze the OSBS reaction, this family is extraordinarily divergent, with some members sharing <15% identity. In addition, a member of this family, Amycolatopsis OSBS/NAAAR, is promiscuous, catalyzing both dehydration and racemization. Although the OSBS/NAAAR family appears to have a single evolutionary origin, no sequence or structural motifs unique to this family could be identified; all residues conserved in the family are also found in enolase superfamily members that have different functions. Based on their species distribution, several uncharacterized proteins similar to Amycolatopsis OSBS/NAAAR appear to have been transmitted by lateral gene transfer. Like Amycolatopsis OSBS/NAAAR, these might have additional or alternative functions to OSBS because many are from organisms lacking the pathway in which OSBS is an intermediate. In addition to functional differences, the OSBS/NAAAR family exhibits surprising structural variations, including large differences in orientation between the two domains. These results offer several insights into protein evolution. First, orthologous proteins can exhibit significant structural variation, and specificity can be maintained with little conservation of ligand-contacting residues. Second, the discovery of a set of proteins similar to Amycolatopsis OSBS/NAAAR supports the hypothesis that new protein functions evolve through promiscuous intermediates. Finally, a combination of evolutionary, structural, and sequence analyses identified characteristics that might prime proteins, such as Amycolatopsis OSBS/NAAAR, for the evolution of new activities.     

The tomato early fruit specific gene <Emphasis Type="Italic">Lefsm1</Emphasis> defines a novel class of plant-specific SANT/MYB domain proteins

We describe here a novel plant-specific gene, Lefsm1 (fruit SANT/MYB-like 1) harboring a single SANT/MYB domain. The expression of Lefsm1 is specific to the very early stages of tomato (Lycopersicon esculentum) fruit development. Ectopic expression of Lefsm1 results in severe developmental alterations manifested in retarded growth, and reduced apical dominance during tomato and Arabidopsis seedling development. A promoter sequence residing 1.0 kb upstream to the translation initiation codon confers the organ-specific expression of the gene. Lefsm1 belongs to a novel small gene family consisting of five to six members in tomato, Arabidopsis and rice. The SANT/MYB domain of LeFSM1 and its orthologs in Arabidopsis and rice differs from that of all other plant or animal MYB proteins and from the SANT domains found in part of the chromatin remodeling proteins. Together, our results indicate that Lefsm1 is a founding member of a small family of proteins containing a novel MYB/SANT domain which is likely to participate in the regulation of a plant-specific developmental program.     

Molecular and cellular characterisation of LjAMT2;1, an ammonium transporter from the model legume Lotus japonicus

Two related families of ammonium transporters have been identified and partially characterised in plants in the past; the AMT1 and AMT2 families. Most attention has focused on the larger of the two families, the AMT1 family, which contains members that are likely to fulfil different, possibly overlapping physiological roles in plants, including uptake of ammonium from the soil. The possible physiological functions of AMT2 proteins are less clear. Lack of data on cellular and tissue location of gene expression, and the intracellular location of proteins limit our understanding of the physiological role of all AMT proteins. We have cloned the first AMT2 family member from a legume, LjAMT2;1 of Lotus japonicus, and demonstrated that it functions as an ammonium transporter by complementing a yeast mutant defective in ammonium uptake. However, like AtAMT2 from Arabidopsis, and unlike AMT1 transporters from several plant species, LjAMT2;1 was unable to transport methylammonium. The LjAMT2;1 gene was found to be expressed constitutively throughout Lotus plants. In situ RNA hybridisation revealed LjAMT2;1 expression in all major tissues of nodules. Transient expression of LjAMT2;1-GFP fusion protein in plant cells indicated that the transporter is located on the plasma membrane. In view of the fact that nodules derive ammonium internally, rather than from the soil, the results implicate LjAMT2;1 in the recovery of ammonium lost from nodule cells by efflux. A similar role may be fulfilled in other organs, especially leaves, which liberate ammonium during normal metabolism.