The significance of PTEN's protein phosphatase activity

Many hundreds of research papers over the last ten years have established the significance of PTEN's lipid phosphatase activity in mediating many of its effects on specific cellular processes in many different cell types, including cell growth, proliferation, survival, and migration ([Backman et al., 2002], [Iijima et al., 2002], [Leslie and Downes, 2002] and [Salmena et al., 2008]). In some cases, detailed signalling mechanisms have been identified by which these PtdInsP₃-dependent effects are manifest ([Kolsch et al., 2008], [Manning and Cantley, 2007] and [Tee and Blenis, 2005]). Further, in some settings, in vivo data from, for example genetic deletion of PTEN, relates closely with independent manipulation of the PI3K/Akt signalling pathway ([Bayascas et al., 2005], [Chen et al., 2006], [Crackower et al., 2002] and [Ma et al., 2005]). Together these studies indicate that the dominant effects of PTEN function are mediated through its regulation of PtdInsP₃-dependent signalling, but that its protein phosphatase activity also contributes in some settings. These conclusions are of great importance given the intense efforts underway to develop PI3K (EC 2.7.1.153) inhibitors as cancer therapeutics. The experiments reviewed here have firmly established that the protein phosphatase activity of PTEN plays a role in the regulation of cellular processes including migration. On the other hand, it has not been established beyond doubt that PTEN acts on substrates other than itself; no such substrates have been confidently identified and effector mechanisms for PTEN's protein phosphatase activity are currently unclear. The goal for future research must be firstly to understand the signalling mechanisms by which PTEN protein phosphatase activity acts: whether this is through identifying substrates, or working out how autodephosphorylation mediates its effects. Secondly, and critically, the significance of PTEN's protein phosphatase activity must be established in vivo. This can be achieved through relating the phenotypes intervening with both PTEN and with protein phosphatase effector pathways when they are identified, and through the generation of mouse models expressing substrate selective PTEN mutants. We should then be able to answer the important question of whether PTEN's protein phosphatase activity contributes to tumour suppression.     

Phosphatidic acid phosphohydrolase in the regulation of inflammatory signaling

Since the cloning and characterization of PAP-1 by Carman's group, a preponderance of new information has emerged pertaining to the molecular function of the enzyme and its physiological function in lipid metabolism ([Carman and Han, 2006] and [Donkor et al., 2007]). As a consequence of this development, PAP-1 and lipin research have informed us further regarding lipid metabolism and adipose tissue development ([Carman and Han, 2006] and [Reue and Zhang, 2008]). In recent years, the field of inflammatory lipid signaling has undergone a great expansion and has come to identify a wide variety of protein and metabolic inflammatory mediators, including PAP-1 as well as PLD, PKC, PLC and DAG. The focus of this review was to summarize experimental evidence supporting a role for PAP-1 in inflammatory signaling, which is summarized in Scheme 2 (Grkovich et al., in press). Further clarification is needed to identify the precise signaling functions and downstream targets of DAG forming enzymes, such as PLD/PAP-1 and PLC, as evidence supports both as participants in inflammatory signaling in different systems. Clarification of these regulatory questions should lead to a more complete understanding of inflammatory signaling while helping to identify future pharmacological targets.     

Polarity proteins regulate mammalian cell–cell junctions and cancer pathogenesis

Polarity pathways regulate important functions during the formation and maintenance of cell–cell junctions and during morphogenesis. In addition, cell polarity pathways are emerging as critical regulators of initiation and progression of carcinoma by functioning as tumor suppressors, downstream of oncogenes, or promoters of the metastatic process (Figure 2). It is highly likely that further analysis of cell polarity proteins and the pathways they control will identify novel biomarkers and potential drug targets for managing and treating patients with carcinoma.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

Identification of novel VHL regulated genes by transcriptomic analysis of RCC10 renal carcinoma cells

Previous microarray studies have revealed a broad range of genes which are regulated by VHL and have provided much insight into how VHL may function as a tumour suppressor gene ([Wykoff et al., 2000b] and [Zatyka et al., 2002]). The current study has highlighted several genes of interest which are not currently recognised as being regulated by VHL. Of the candidate VHL regulated genes that we identified ASS was selected for further study due to its therapeutic implications. Tumours with low ASS levels display a reduced capacity to synthesise arginine, and as such are reliant on extracellular arginine for normal cellular function. Promising results in mouse xenograft models have shown that arginine deprivation may be a useful treatment strategy for these tumours. Understanding how ASS expression levels are regulated should provide insight into which tumour types would be most sensitive to treatment with arginine degrading enzymes. In this study we provide strong evidence that VHL status regulates ASS expression levels in three independent CCRCC cell backgrounds. Regulation of ASS by VHL/HIF suggests that arginine deprivation may be useful in the treatment of VHL defective CCRCCs and non-renal hypoxic tumours.     

The Conserved miR-51 microRNA Family Is Redundantly Required for Embryonic Development and Pharynx Attachment in Caenorhabditis elegans

A major question about cytokinesis concerns the role of the septin proteins, which localize to the division site in all animal and fungal cells but are essential for cytokinesis only in some cell types. For example, in Schizosaccharomyces pombe, four septins localize to the division site, but deletion of the four genes produces only a modest delay in cell separation. To ask if the S. pombe septins function redundantly in cytokinesis, we conducted a synthetic-lethal screen in a septin-deficient strain and identified seven mutations. One mutation affects Cdc4, a myosin light chain that is an essential component of the cytokinetic actomyosin ring. Five others cause frequent cell lysis during cell separation and map to two loci. These mutations and their dosage suppressors define a signaling pathway (including Rho1 and a novel arrestin) for repairing cell-wall damage. The seventh mutation affects the poorly understood RNA-binding protein Scw1 and severely delays cell separation when combined either with a septin mutation or with a mutation affecting the septin-interacting, anillin-like protein Mid2, suggesting that Scw1 functions in a pathway parallel to that of the septins. Taken together, our results suggest that the S. pombe septins participate redundantly in one or more pathways that cooperate with the actomyosin ring during cytokinesis and that a septin defect causes septum defects that can be repaired effectively only when the cell-integrity pathway is intact.THE fission yeast Schizosaccharomyces pombe provides an outstanding model system for studies of cytokinesis (McCollum and Gould 2001; Balasubramanian et al. 2004; Pollard and Wu 2010). As in most animal cells, successful cytokinesis in S. pombe requires an actomyosin ring (AMR). The AMR begins to assemble at the G₂/M transition and involves the type II myosin heavy chains Myo2 and Myp2 and the light chains Cdc4 and Rlc1 (Wu et al. 2003). Myo2 and Cdc4 are essential for cytokinesis under all known conditions, Rlc1 is important at all temperatures but essential only at low temperatures, and Myp2 is essential only under stress conditions. As the AMR constricts, a septum of cell wall is formed between the daughter cells. The primary septum is sandwiched by secondary septa and subsequently digested to allow cell separation (Humbel et al. 2001; Sipiczki 2007). Because of the internal turgor pressure of the cells, the proper assembly and structural integrity of the septal layers are essential for cell survival.Septum formation involves the β-glucan synthases Bgs1/Cps1/Drc1, Bgs3, and Bgs4 (Ishiguro et al. 1997; Le Goff et al. 1999; Liu et al. 1999, 2002; Martín et al. 2003; Cortés et al. 2005) and the α-glucan synthase Ags1/Mok1 (Hochstenbach et al. 1998; Katayama et al. 1999). These synthases are regulated by the Rho GTPases Rho1 and Rho2 and the protein kinase C isoforms Pck1 and Pck2 (Arellano et al. 1996, 1997, 1999; Nakano et al. 1997; Hirata et al. 1998; Calonge et al. 2000; Sayers et al. 2000; Ma et al. 2006; Barba et al. 2008; García et al. 2009b). The Rho GTPases themselves appear to be regulated by both GTPase-activating proteins (GAPs) and guanine-nucleotide-exchange factors (GEFs) (Nakano et al. 2001; Calonge et al. 2003; Iwaki et al. 2003; Tajadura et al. 2004; Morrell-Falvey et al. 2005; Mutoh et al. 2005; García et al. 2006, 2009a,b). In addition, septum formation and AMR function appear to be interdependent. In the absence of a normal AMR, cells form aberrant septa and/or deposit septal materials at random locations, whereas a mutant defective in septum formation (bgs1) is also defective in AMR constriction (Gould and Simanis 1997; Le Goff et al. 1999; Liu et al. 1999, 2000). Both AMR constriction and septum formation also depend on the septation initiation network involving the small GTPase Spg1 (McCollum and Gould 2001; Krapp and Simanis 2008). Despite this considerable progress, many questions remain about the mechanisms and regulation of septum formation and its relationships to the function of the AMR.One major question concerns the role(s) of the septins. Proteins of this family are ubiquitous in fungal and animal cells and typically localize to the cell cortex, where they appear to serve as scaffolds and diffusion barriers for other proteins that participate in a wide variety of cellular processes (Longtine et al. 1996; Gladfelter et al. 2001; Hall et al. 2008; Caudron and Barral 2009). Despite the recent progress in elucidating the mechanisms of septin assembly (John et al. 2007; Sirajuddin et al. 2007; Bertin et al. 2008; McMurray and Thorner 2008), the details of septin function remain obscure. However, one prominent role of the septins and associated proteins is in cytokinesis. Septins concentrate at the division site in every cell type that has been examined, and in Saccharomyces cerevisiae (Hartwell 1971; Longtine et al. 1996; Lippincott et al. 2001; Dobbelaere and Barral 2004) and at least some Drosophila (Neufeld and Rubin 1994; Adam et al. 2000) and mammalian (Kinoshita et al. 1997; Surka et al. 2002) cell types, the septins are essential for cytokinesis. In S. cerevisiae, the septins are required for formation of the AMR (Bi et al. 1998; Lippincott and Li 1998). However, this cannot be their only role, because the AMR itself is not essential for cytokinesis in this organism (Bi et al. 1998; Korinek et al. 2000; Schmidt et al. 2002). Moreover, there is no evidence that the septins are necessary for AMR formation or function in any other organism. A further complication is that in some cell types, including most Caenorhabditis elegans cells (Nguyen et al. 2000; Maddox et al. 2007) and some Drosophila cells (Adam et al. 2000; Field et al. 2008), the septins do not appear to be essential for cytokinesis even though they localize to the division site.S. pombe has seven septins, four of which (Spn1, Spn2, Spn3, and Spn4) are expressed in vegetative cells and localize to the division site shortly before AMR constriction and septum formation (Longtine et al. 1996; Berlin et al. 2003; Tasto et al. 2003; Wu et al. 2003; An et al. 2004; Petit et al. 2005; Pan et al. 2007; Onishi et al. 2010). Spn1 and Spn4 appear to be the core members of the septin complex (An et al. 2004; McMurray and Thorner 2008), and mutants lacking either of these proteins do not assemble the others at the division site. Assembly of a normal septin ring also depends on the anillin-like protein Mid2, which colocalizes with the septins (Berlin et al. 2003; Tasto et al. 2003). Surprisingly, mutants lacking the septins are viable and form seemingly complete septa with approximately normal timing. These mutants do, however, display a variable delay in separation of the daughter cells, suggesting that the septins play some role(s) in the proper completion of the septum or in subsequent processes necessary for cell separation (Longtine et al. 1996; An et al. 2004; Martín-Cuadrado et al. 2005).It is possible that the septins localize to the division site and yet are nonessential for division in some cell types because their role is redundant with that of some other protein(s) or pathway(s). To explore this possibility in S. pombe, we screened for mutations that were lethal in combination with a lack of septins. The results suggest that the septins cooperate with the AMR during cytokinesis and that, in the absence of septin function, the septum is not formed properly, so that an intact system for recognizing and repairing cell-wall damage becomes critical for cell survival.     

VTC4 Is a Bifunctional Enzyme That Affects Myoinositol and Ascorbate Biosynthesis in Plants

Myoinositol synthesis and catabolism are crucial in many multiceullar eukaryotes for the production of phosphatidylinositol signaling molecules, glycerophosphoinositide membrane anchors, cell wall pectic noncellulosic polysaccharides, and several other molecules including ascorbate. Myoinositol monophosphatase (IMP) is a major enzyme required for the synthesis of myoinositol and the breakdown of myoinositol (1,4,5)trisphosphate, a potent second messenger involved in many biological activities. It has been shown that the VTC4 enzyme from kiwifruit (Actinidia deliciosa) has similarity to IMP and can hydrolyze l-galactose 1-phosphate (l-Gal 1-P), suggesting that this enzyme may be bifunctional and linked with two potential pathways of plant ascorbate synthesis. We describe here the kinetic comparison of the Arabidopsis (Arabidopsis thaliana) recombinant VTC4 with d-myoinositol 3-phosphate (d-Ins 3-P) and l-Gal 1-P. Purified VTC4 has only a small difference in the V_max/K_m for l-Gal 1-P as compared with d-Ins 3-P and can utilize other related substrates. Inhibition by either Ca²⁺ or Li⁺, known to disrupt cell signaling, was the same with both l-Gal 1-P and d-Ins 3-P. To determine whether the VTC4 gene impacts myoinositol synthesis in Arabidopsis, we isolated T-DNA knockout lines of VTC4 that exhibit small perturbations in abscisic acid, salt, and cold responses. Analysis of metabolite levels in vtc4 mutants showed that less myoinositol and ascorbate accumulate in these mutants. Therefore, VTC4 is a bifunctional enzyme that impacts both myoinositol and ascorbate synthesis pathways.Myoinositol is a six-member carbon ring polyol that is synthesized by both eukaryotes and prokaryotes (for review, see Michell, 2007). In multiceullar eukaryotes, myoinositol becomes incorporated into many crucial cellular compounds, including those involved in signal transduction such as phosphatidylinositol phosphates and myoinositol phosphates (InsPs; for review, see Boss et al., 2006), gene expression (InsPs; for review, see Alcazar-Roman and Wente, 2007), auxin perception and phosphorus storage (myoinositol hexakisphosphate [InsP₆]; for review, see Raboy and Bowen, 2006; Tan et al., 2007), membrane tethering (glycerophosphoinositide anchors; for review, see Fujita and Jigami, 2007), stress tolerance (ononitol, pinitol; for review, see Taji et al., 2006), and oligosaccharide synthesis (galactinol; for review, see Karner et al., 2004; Fig. 1). Its primary breakdown product, d-GlcUA, is utilized for the synthesis of cell wall pectic noncellulosic compounds (for review, see Loewus, 2006) and, in some organisms, ascorbate (for review, see Linster and Van Schaftingen, 2007). Thus, myoinositol synthesis and catabolism affect metabolites involved in many different and critical biochemical pathways.Open in a separate windowFigure 1.Myoinositol synthesis and metabolism pathway. De novo synthesis of myoinositol (i.e. the Loewus pathway) is catalyzed by myoinositol phosphate synthase (MIPS) and IMP, where its immediate precursor is d-Ins 3-P = l-Ins 1-P. IMP also regenerates myoinositol from the second messenger d-Ins(1,4,5)P₃. Oxidation of inositol by myoinositol oxygenase (MIOX) produces d-GlcUA (d-GlcA), which is a possible entry point into ascorbate synthesis. The major route to ascorbate in plants is the Smirnoff-Wheeler pathway and utilizes GDP-d-Man. VTC4 has homology to the animal IMPs and has been shown to catalyze the conversion of l-Gal 1-P to l-Gal in the Smirnoff-Wheeler pathway. Inositol is also the precursor for the synthesis of several compounds indicated in gray. The asterisk indicates the inositol signaling pathway.Although organisms incorporate myoinositol into various compounds, there is only one biosynthetic route to produce myoinositol in what has been referred to as the Loewus pathway (Eisenberg et al., 1964; Chen and Charalampous, 1966; Sherman et al., 1969; Loewus and Loewus, 1980; Loewus et al., 1980). The conversion of Glc-6-P to InsP is catalyzed by the myoinositol phosphate synthase (EC 5.5.1.4; for review, see GhoshDastidar et al., 2006). The product of this reaction can be referred to as either l-myoinositol 1-P or d-myoinositol 3-P (d-Ins 3-P), which are equivalent compounds. The conversion of d-Ins 3-P to free myoinositol is catalyzed by the myoinositol monophosphatase (IMP; EC 3.1.3.25; for review, see Torabinejad and Gillaspy, 2006). We have been interested in the function of IMP in both de novo myoinositol synthesis and during myoinositol second messenger recycling from myoinositol phosphate signaling molecules, such as d-myoinositol 1-P (d-Ins 1-P; Fig. 1). IMP is encoded by multiple genes in plants (e.g. three IMP genes have been examined in tomato [Solanum lycopersicum]; Gillaspy et al., 1995). The three different tomato IMPs are highly conserved enzymes that act specifically on monophosphorylated substrates and are inhibited by LiCl (Gillaspy et al., 1995; Berdy et al., 2001). IMP gene expression is developmentally regulated, as is the accumulation of IMP proteins, with maximal levels being present in plant tissues undergoing rapid cell divisions, such as seedlings and developing anthers (Gillaspy et al., 1995; Suzuki et al., 2007).In contrast, Arabidopsis (Arabidopsis thaliana) contains one potential IMP gene (At3g02870), which was previously identified as functioning in ascorbate synthesis and named VTC4 (Laing et al., 2004; Conklin et al., 2006). Two other genes (At1g31190 and At4g39120) encode proteins that we have classified as IMP-like (IMPL), because of their greater homology to the prokaryotic IMPs, such as the SuhB (Matsuhisa et al., 1995; Chen and Roberts, 2000) and CysQ (Neuwald et al., 1992; Peng and Verma, 1995) proteins. Prokaryotic IMPLs are known to dephosphorylate d-Ins 1-P and other substrates in vitro; however, the function of these IMPL proteins is currently unknown (for review, see Roberts, 2006).Intriguing data suggest that animal IMP is a bifunctional enzyme. The animal IMP hydrolyzes d-Gal 1-P, which is involved in Gal metabolism (Parthasarathy et al., 1997). Furthermore, expression of human IMP can suppress Gal toxicity in yeast (Mehta et al., 1999). Efforts to isolate an l-Gal 1-P phosphatase required for ascorbate synthesis in plants revealed that the kiwi (Actinidia deliciosa) and Arabidopsis VTC4 can hydrolyze l-Gal 1-P (Laing et al., 2004). This fact prompted the proposal that VTC4 functions mainly to hydrolyze l-Gal 1-P during ascorbate synthesis and that other, unidentified enzymes might be responsible for de novo myoinositol synthesis in plants. This idea is supported by the fact that a vtc4 loss-of-function mutant contains lower ascorbate levels (Conklin et al., 2006).Since VTC4 and the IMPLs are the best candidates for enzymes with IMP activity, it is crucial to understand whether these enzymes impact myoinositol synthesis in plants in vivo. To determine whether VTC4 is bifunctional and functions during InsP hydrolysis as well as l-Gal 1-P hydrolysis, we expressed recombinant Arabidopsis VTC4 protein and compared the kinetic constants for both d-Ins 3-P and l-Gal 1-P. In contrast to previously reported results, we report here that VTC4 hydrolyzes both substrates well and thus should be considered a bifunctional enzyme. We investigated loss-of-function vtc4 mutant plants and confirm that these plants contain lower ascorbate levels. We also find reduced myoinositol levels in vtc4 mutants, supporting a direct role for VTC4 in InsP hydrolysis in plants.     

Growth of Transplastomic Cells Expressing d-Amino Acid Oxidase in Chloroplasts Is Tolerant to d-Alanine and Inhibited by d-Valine

Dual-conditional positive/negative selection markers are versatile genetic tools for manipulating genomes. Plastid genomes are relatively small and conserved DNA molecules that can be manipulated precisely by homologous recombination. High-yield expression of recombinant products and maternal inheritance of plastid-encoded traits make plastids attractive sites for modification. Here, we describe the cloning and expression of a dao gene encoding d-amino acid oxidase from Schizosaccharomyces pombe in tobacco (Nicotiana tabacum) plastids. The results provide genetic evidence for the uptake of d-amino acids into plastids, which contain a target that is inhibited by d-alanine. Importantly, this nonantibiotic-based selection system allows the use of cheap and widely available d-amino acids, which are relatively nontoxic to animals and microbes, to either select against (d-valine) or for (d-alanine) cells containing transgenic plastids. Positive/negative selection with d-amino acids was effective in vitro and against transplastomic seedlings grown in soil. The dual functionality of dao is highly suited to the polyploid plastid compartment, where it can be used to provide tolerance against potential d-alanine-based herbicides, control the timing of recombination events such as marker excision, influence the segregation of transgenic plastid genomes, identify loci affecting dao function in mutant screens, and develop d-valine-based methods to manage the spread of transgenic plastids tagged with dao.Selectable marker genes provide powerful genetic tools for manipulating plant genomes (Miki and McHugh, 2004; Day and Goldschmidt-Clermont, 2011). High and stable accumulation of high-value products in transgenic plastids (Daniell et al., 2009; Maliga and Bock, 2011), combined with restricted dissemination of plastid transgenes in pollen (Daniell et al., 1998; Ruf et al., 2007; Svab and Maliga, 2007), make plastid genomes an attractive target for modification. Reverse genetics allows site-directed changes to be introduced into the important set of genes present in plastids to study and ultimately improve their function (Whitney et al., 2011; Day, 2012). Positive conditional selectable marker genes allow transgenic cells to divide in the presence of chemicals such as antibiotics or herbicides that inhibit wild-type untransformed cells and underpin the development of herbicide-resistant transplastomic crops (Daniell et al., 1998, 2009; Iamtham and Day, 2000; Lutz et al., 2001; Ye et al., 2001; Dufourmantel et al., 2007; Shimizu et al., 2008). Negative conditional selectable marker genes inhibit the proliferation of transgenic cells when exposed to compounds that have a limited impact on the viability of wild-type cells (Miki and McHugh, 2004). Most commonly, the enzyme product of a negative selection gene converts an exogenous chemical substrate into a toxic product. Negative selectable markers allow genetic restriction technologies to manage the spread of transgenic crops (Daniell, 2002; Hills et al., 2007) and are valuable components of genome manipulation technologies involving recombination (Hardy et al., 2010). Negative selection can be used to promote genome changes by, for example, linking recombination events to the excision of a negative selection marker gene. Dual positive/negative selectable marker genes are particularly versatile tools for genome engineering. An example is the uracil3 (URA3) gene marker in Saccharomyces cerevisiae, where selection for or against URA3 encoding orotidine 5''-phosphate decarboxylase has been used to influence the timing of recombination events to manipulate chromosomes and introduce site-directed mutations into S. cerevisiae genes (Boeke et al., 1987).Homologous recombination is an effective tool for genome engineering in bacteria (Link et al., 1997) and S. cerevisiae (Boeke et al., 1987) and is the predominant recombination pathway operating in plastids (Day and Madesis, 2007). Plastid genomes are relatively small, sequenced in over 200 species (Jansen and Ruhlman, 2012), polyploid, and are highly suitable targets for genome engineering (O’Neill et al., 2012). Establishing a dual-marker system in plastids would allow control over the timing and efficiency of homologous recombination events, such as marker excision between direct repeats (Iamtham and Day, 2000). New positive selection markers not involving antibiotics are candidates for developing herbicide-tolerant transplastomic crops, contained by maternal inheritance of plastids (Daniell et al., 1998). Genetic containment methods require the insertion of a conditional negative selectable marker into the plastid genome and may be desirable in transplastomic “pharma” crops and cells expressing products for medicine (Daniell et al., 2009; Oey et al., 2009a, 2009b; Ruhlman et al., 2010; Gisby et al., 2011). Negative markers inserted into the nucleus would be ineffective for preventing the spread of transgenic plastids because nuclear and chloroplast genes are inherited independently and by different mechanisms.Nuclear expression of the Rhodotorula gracilis dao gene encoding d-amino acid oxidase (DAAO; EC 1.4.3.3) allows positive or negative selection of transgenic plant cells (Erikson et al., 2004). Transgenic nuclear dao plants were tolerant to d-Ala and d-Ser but sensitive to d-Val and d-Ile. A reversal of the tolerance pattern was observed in wild-type plants, which were sensitive to d-Ala and d-Ser but tolerant to d-Val and d-Ile. The toxic ketoacids 3-methyl-2-oxobutanoate and 3-methyl-2-oxopentanoate are produced by DAAO-catalyzed deamination of d-Val and d-Ile, respectively (Erikson et al., 2004). Originally developed in Arabidopsis (Arabidopsis thaliana), the system has been applied to crops (Lai et al., 2007). Importantly, d-amino acids are relatively cheap and nontoxic (Gullino et al., 1956; Friedman, 1999), thereby facilitating their use outside the laboratory. Cost and safety are important factors for applications as positive selection herbicides to control weeds in transgenic crops and negative selection agents to control the spread of transgenic crops. A dual selectable marker gene has not previously been described in plastids. The bacterial codA gene, encoding cytosine deaminase (Mullen et al., 1992), is the sole negative selection marker shown to work in plastids (Serino and Maliga, 1997). Transplastomic cells expressing codA are sensitive to the antibiotic 5-fluorocytosine (Serino and Maliga, 1997), which is used to treat fungal infections in humans, with possible side effects including hepatotoxicity (Steer et al., 1972). The relatively high cost of 5-fluorocytosine and its potential toxicity on nontarget organisms would hinder its use as a negative selection agent to manage the spread of transgenic crops expressing codA.Plastids are important centers for amino acid synthesis and metabolism (Lancien et al., 2006), but the impact of d-amino acids on plastids had not been reported previously. Here, we have developed a plastid marker based on the Schizosaccharomyces pombe dao gene (Wood et al., 2002) that confers either positive (d-Ala) or negative (d-Val) selection on transplastomic cells. Our results are consistent with d-amino acids being transported into plastids, where d-Ala inhibits one or more plastid functions and d-Val is converted into a toxic product by DAAO. The plastid dao gene provides a new versatile marker for plastid genetics, with applications for developing d-Ala-tolerant transplastomic plants, d-Val-based containment of transgenic plastids, and controlling the timing of recombination events in plastid genomes.     

Cloning and Characterization of AtRGP1 : A Reversibly Autoglycosylated Arabidopsis Protein Implicated in Cell Wall Biosynthesis

A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsis thaliana, Reversibly Glycosylated Polypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologs in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells. Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes. We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.The primary cell wall of dicot plants is laid down by young cells prior to the cessation of elongation and secondary wall deposition. Making up to 90% of the cell''s dry weight, the extracellular matrix is important for many processes, including morphogenesis, growth, disease resistance, recognition, signaling, digestibility, nutrition, and decay. The composition of the cell wall has been extensively described (Bacic et al., 1988; Levy and Staehelin, 1992; Zablackis et al., 1995), and yet many questions remain unanswered regarding the synthesis and interaction of these components to provide cells with a functional wall (Carpita and Gibeaut, 1993; Carpita et al., 1996).Heteropolysaccharide biosynthesis can be divided into four steps: (a) chain or backbone initiation, (b) elongation, (c) side-chain addition, and (d) termination and extracellular deposition (Waldron and Brett, 1985). The similarity between various polysaccharide backbones leads to the prediction that the synthesizing machinery would be conserved between them. For example, the backbone of xyloglucan polymers, β-1,4 glucan, can be synthesized independently of or concurrently with side-chain addition (Campbell et al., 1988; White et al., 1993), and this polymer and the chains that make up cellulose are identical. The later addition of side chains to xyloglucan are catalyzed by specific transferases (Kleene and Berger, 1993) such as xylosyltransferase (Campbell et al., 1988), galactosyltransferase, and fucosyltransferase (Faïk et al., 1997), all of which are localized to the Golgi compartment (Brummell et al., 1990; Driouich et al., 1993; Staehelin and Moore, 1995).The enzymes involved in wall biosynthesis have been recalcitrant to isolation (Carpita et al., 1996; Albersheim et al., 1997). Only recently has the first gene encoding putative cellulose biosynthetic enzymes, celA, been isolated from cotton (Gossypium hirsutum) and rice (Oryza sativa; Pear et al., 1996).During studies of polysaccharide synthesis in pea (Pisum sativum) Golgi membranes, Dhugga et al. (1991) identified a 41-kD protein doublet that they suggested was involved in polysaccharide synthesis. The authors showed that this protein could be glycosylated by radiolabeled UDP-Glc but that this labeling could be reversibly competed with by unlabeled UDP-Glc, UDP-Xyl, and UDP-Gal, the sugars that make up xyloglucan (Hayashi, 1989). The 41-kD protein was named PsRGP1 (P. sativum Reversibly Glycosylated Polypeptide-1; Dhugga et al., 1997). Furthermore, the conditions that stimulate or inhibit Golgi-localized β-glucan synthase activity are the same conditions that stimulate or inhibit the glycosylation of PsRGP1 (Dhugga et al., 1991). To address the role of this protein in polysaccharide synthesis, the authors purified the polypeptides and obtained the sequences from tryptic peptides (Dhugga and Ray, 1994). Antibodies raised against PsRGP1 showed that it is soluble and localized to the plasma membrane (Dhugga et al., 1991) and Golgi compartment (Dhugga et al., 1997). In addition to its Golgi localization, the steady-state glycosylation of PsRGP1 is approximately 10:7:3 (UDP-Glc:-Xyl:-Gal), which is similar to the typical sugar composition of xyloglucan (1.0:0.75:0.25; Dhugga et al., 1997).We were interested in studying various aspects of cell wall metabolism, including the synthesis of polysaccharides and their delivery to the cell wall. Studies in pea have shown that a 41-kD protein may be involved in cell wall polysaccharide synthesis, possibly that of xyloglucan (Dhugga et al., 1997). Here we report the characterization of AtRGP1 (Arabidopsis thaliana Reversibly Glycosylated Polypeptide-1), a soluble protein that can also be found weakly associated with membrane fractions, most likely the Golgi fraction. The reversible nature of the glycosylation of this Arabidopsis homolog by the substrates used to make polysaccharides (nucleotide sugars) suggests a possible role for AtRGP1 in polysaccharide biosynthesis.     

Genetic Modifiers of dFMR1 Encode RNA Granule Components in Drosophila

Mechanisms of neuronal mRNA localization and translation are of considerable biological interest. Spatially regulated mRNA translation contributes to cell-fate decisions and axon guidance during development, as well as to long-term synaptic plasticity in adulthood. The Fragile-X Mental Retardation protein (FMRP/dFMR1) is one of the best-studied neuronal translational control molecules and here we describe the identification and early characterization of proteins likely to function in the dFMR1 pathway. Induction of the dFMR1 in sevenless-expressing cells of the Drosophila eye causes a disorganized (rough) eye through a mechanism that requires residues necessary for dFMR1/FMRP''s translational repressor function. Several mutations in dco, orb2, pAbp, rm62, and smD3 genes dominantly suppress the sev-dfmr1 rough-eye phenotype, suggesting that they are required for dFMR1-mediated processes. The encoded proteins localize to dFMR1-containing neuronal mRNPs in neurites of cultured neurons, and/or have an effect on dendritic branching predicted for bona fide neuronal translational repressors. Genetic mosaic analyses indicate that dco, orb2, rm62, smD3, and dfmr1 are dispensable for translational repression of hid, a microRNA target gene, known to be repressed in wing discs by the bantam miRNA. Thus, the encoded proteins may function as miRNA- and/or mRNA-specific translational regulators in vivo.THE subcellular localization and regulated translation of stored mRNAs contributes to cellular asymmetry and subcellular specialization (Lecuyer et al. 2007; Martin and Ephrussi 2009). In mature neurons, local protein synthesis at active synapses may contribute to synapse-specific plasticity that underlies persistent forms of memory (Casadio et al. 1999; Ashraf et al. 2006; Sutton and Schuman 2006; Richter and Klann 2009). During this process, synaptic activity causes local translation of mRNAs normally stored in translationally repressed synaptic mRNPs (Sutton and Schuman 2006; Richter and Klann 2009). While specific neuronal translational repressors and microRNAs have been implicated in this process, their involvement in local translation that underlies memory, as well as the underlying mechanisms, are generally not well understood (Schratt et al. 2006; Keleman et al. 2007; Kwak et al. 2008; Li et al. 2008; Richter and Klann 2009). Furthermore, it remains possible that there are neuron-specific, mRNA-specific, and stimulus-pattern specific pathways for neuronal translational control (Raab-Graham et al. 2006; Giorgi et al. 2007).The Fragile-X Mental Retardation protein (FMRP) is among the best studied of neuronal translational repressors, in part due to its association with human neurodevelopmental disease (Pieretti et al. 1991; Mazroui et al. 2002; Gao 2008). Consistent with function in synaptic translation required for memory formation, mutations in FMRP are associated with increased synaptic translation, enhanced LTD, increased synapse growth, and also with enhanced long-term memory (Zhang et al. 2001; Huber et al. 2002; Bolduc et al. 2008; Dictenberg et al. 2008).FMRP co-immunoprecipitates with components of the RNAi and miRNA machinery and appears to be required for aspects of miRNA function in neurons (Caudy et al. 2002; Ishizuka et al. 2002; Jin et al. 2004b; Gao 2008). In addition, FMRP associates with neuronal polyribosomes as well as with Staufen-containing ribonucleoprotein (mRNP) granules easily observed in neurites of cultured neurons (Feng et al. 1997; Krichevsky and Kosik 2001; Mazroui et al. 2002; Kanai et al. 2004; Barbee et al. 2006; Bramham and Wells 2007; Bassell and Warren 2008; Dictenberg et al. 2008). FMRP-containing neuronal mRNPs contain not only several ubiquitous translational control molecules, but also CaMKII and Arc mRNAs, whose translation is locally controlled at synapses (Rook et al. 2000; Krichevsky and Kosik 2001; Kanai et al. 2004; Barbee et al. 2006). Thus, FMRP-containing RNA particles are probably translationally repressed and transported along microtubules from the neuronal cell body to synaptic sites in dendrites where local synaptic activity can induce their translation (Kiebler and Bassell 2006; Dictenberg et al. 2008).The functions of FMRP/dFMR1 in mRNA localization as well as miRNA-dependent and independent forms of translational control is likely to require several other regulatory proteins. To identify such proteins, we used a previously designed and validated genetic screen (Wan et al. 2000; Jin et al. 2004a; Zarnescu et al. 2005). The overexpression of dFMR1 in the fly eye causes a “rough-eye” phenotype through a mechanism that requires (a) key residues in dFMR1 that mediate translational repression in vitro; (b) Ago1, a known components of the miRNA pathway; and (c) a DEAD-box helicase called Me31B, which is a highly conserved protein from yeast (Dhh1p) to humans (Rck54/DDX6) functioning in translational repression and present on neuritic mRNPs (Wan et al. 2000; Laggerbauer et al. 2001; Jin et al. 2004a; Coller and Parker 2005; Barbee et al. 2006; Chu and Rana 2006). To identify other Me31B-like translational repressors and neuronal granule components, we screened mutations in 43 candidate proteins for their ability to modify dFMR1 induced rough-eye phenotype. We describe the results of this genetic screen and follow up experiments to address the potential cellular functions of five genes identified as suppressors of sev-dfmr1.