Luisa Maria Lois

Structures of the SUMO E1 provide mechanistic insights into SUMO activation and E2 recruitment to E1

E1 enzymes facilitate conjugation of ubiquitin and ubiquitin‐like proteins through adenylation, thioester transfer within E1, and thioester transfer from E1 to E2 conjugating proteins. Structures of human heterodimeric Sae1/Sae2‐Mg·ATP and Sae1/Sae2‐SUMO‐1‐Mg·ATP complexes were determined at 2.2 and 2.75 Å resolution, respectively. Despite the presence of Mg·ATP, the Sae1/Sae2‐SUMO‐1‐Mg·ATP structure reveals a substrate complex insomuch as the SUMO C‐terminus remains unmodified within the adenylation site and 35 Å from the catalytic cysteine, suggesting that additional changes within the adenylation site may be required to facilitate chemistry prior to adenylation and thioester transfer. A mechanism for E2 recruitment to E1 is suggested by biochemical and genetic data, each of which supports a direct role for the E1 C‐terminal ubiquitin‐like domain for E2 recruitment during conjugation.

Small Ubiquitin-Like Modifier Modulates Abscisic Acid Signaling in Arabidopsis

Post-translational modification of proteins by small polypeptides, such as ubiquitin, has emerged as a common and important mechanism for regulating protein function. Small ubiquitin-like modifier (SUMO) is a small protein that is structurally related to but functionally different from ubiquitin. We report the identification and functional analysis of AtSUMO1, AtSUMO2, and AtSCE1a as components of the SUMO conjugation (sumoylation) pathway in Arabidopsis. In yeast-two hybrid assays, AtSUMO1/2 interacts specifically with a SUMO-conjugating enzyme but not with a ubiquitin-conjugating enzyme. AtSCE1a, the Arabidopsis SUMO-conjugating enzyme ortholog, conjugates SUMO to RanGAP in vitro. AtSUMO1/2 and AtSCE1a colocalize at the nucleus, and AtSUMO1/2 are conjugated to endogenous SUMO targets in vivo. Analysis of transgenic plants showed that overexpression of AtSUMO1/2 does not have any obvious effect in general plant development, but increased sumoylation levels attenuate abscisic acid (ABA)–mediated growth inhibition and amplify the induction of ABA- and stress-responsive genes such as RD29A. Reduction of AtSCE1a expression levels accentuates ABA-mediated growth inhibition. Our results suggest a role for SUMO in the modulation of the ABA signal transduction pathway.

Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli.

An alternative mevalonate‐independent pathway for isoprenoid biosynthesis has been recently discovered in eubacteria (including Escherichia coli) and plant plastids, although it is not fully elucidated yet. In this work, E. coli cells were engineered to utilize exogenously provided mevalonate and used to demonstrate by a genetic approach that branching of the endogenous pathway results in separate synthesis of the isoprenoid building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In addition, the IPP isomerase encoded by the idi gene was shown to be functional in vivo and to represent the only possibility for interconverting IPP and DMAPP in this bacterium.

Evolution of a signalling system that incorporates both redundancy and diversity: Arabidopsis SUMOylation

The reversible post-translational modifier, SUMO (small ubiquitin-related modifier), modulates the activity of a diverse set of target proteins, resulting in important consequences to the cellular machinery. Conjugation machinery charges the processed SUMO so that it can be linked via an isopeptide bond to a target protein. The removal of SUMO moieties from conjugated proteins by isopeptidases regenerates pools of processed SUMOs and unmodified target proteins. The evolutionarily conserved SUMO-conjugating proteins, E1 and E2, recognize a diverse set of Arabidopsis SUMO proteins using them to modify protein substrates. In contrast, the deSUMOylating enzymes differentially recognize the Arabidopsis SUMO proteins, resulting in specificity of the deconjugating machinery. The specificity of the Arabidopsis deSUMOylating enzymes is further diversified by the addition of regulatory domains. Therefore the SUMO proteins, in this signalling system, have evolved to contain information that allows not only redundancy with the conjugation system but also diversity with the deconjugating enzymes.

Incorporation of [2,3-13C2]- and [2,4-13C2]-d-1-Deoxyxylulose into Ubiquinone of Escherichia coli via the Mevalonate-Independent Pathway for Isoprenoid Biosynthesis

Abstract [2,3- 13 C 2 ]- and [2,4- 13 C 2 ]- d -1-deoxyxylulose were synthesized from [2- 13 C]pyruvate and [1- 13 C]- or [2- 13 C]- dl -glyceraldehyde using the enzyme d -1-deoxyxylulose 5-phosphate synthase from Escherichia coli which was overexpressed in this bacterium. These doubly-labeled isoprenoid precursors in the mevalonate independent route were incorporated into the ubiquinone of E. coli . 1 J 13 C/ 13 C coupling constants were respectively found in isoprenic units between carbon atoms derived from C-3 and C-4 of isopentenyl diphosphate using the former labeled precursor or between C-3 and C-2 using the latter, indicating that d -1-deoxyxylulose was incorporated without prior degradation into isoprenoids and confirming that the branched isoprenic skeleton resulted from a rearrangement of the straight chain from carbohydrate precursor.

Bioinformatic and molecular analysis of hydroxymethylbutenyl diphosphate synthase (GCPE) gene expression during carotenoid accumulation in ripening tomato fruit

Carotenoids are plastidic isoprenoid pigments of great biological and biotechnological interest. The precursors for carotenoid production are synthesized through the recently elucidated methylerythritol phosphate (MEP) pathway. Here we have identified a tomato (Lycopersicon esculentum Mill.) cDNA sequence encoding a full-length protein with homology to the MEP pathway enzyme hydroxymethylbutenyl 4-diphosphate synthase (HDS, also called GCPE). Comparison with other plant and bacterial HDS sequences showed that the plant enzymes contain a plastid-targeting N-terminal sequence and two highly conserved plant-specific domains in the mature protein with no homology to any other sequence in the databases. The ubiquitous distribution of HDS-encoding expressed sequence tags (ESTs) in the tomato collections suggests that the corresponding gene is likely expressed throughout the plant. The role of HDS in controlling the supply of precursors for carotenoid biosynthesis was estimated from the bioinformatic and molecular analysis of transcript abundance in different stages of fruit development. No significant changes in HDS gene expression were deduced from the statistical analysis of EST distribution during fruit ripening, when an active MEP pathway is required to support a massive accumulation of carotenoids. RNA blot experiments confirmed that similar transcript levels were present in both the wild-type and carotenoid-depleted yellow ripe (r) mutant fruit independent of the stage of development and the carotenoid composition of the fruit. Together, our results are consistent with a non-limiting role for HDS in carotenoid biosynthesis during tomato fruit ripening.

5-HYDROXYPENTANE-2,3 -DIONE (LAURENCIONE), A BACTERIAL METABOLITE OF 1-DEOXY-D-THREO-PENTULOSE

Abstract Cell-free systems from the bacteria Escherichia coli and Klebsiella planticola that were incubated with 13 C labeled pyruvate and D-glyceraldehyde synthesized 5-hydroxypentane-2,3-dione (laurencione) along with 1-deoxy-D- threo -pentulose (1-deoxy-D-xylulose). Both compounds showed identical labeling patterns, indicating that the C 5 skeletons were derived from the condensation of (hydroxyethyl)thiamin on D-glyceraldehyde. Conversion of [5,5- 2 H 2 ]deoxyxylulose into laurencione by a cell-free system from E. coli showed that the α-dione is obtained from the pentulose by water elimination.

A Simplified and Rapid Method for the Isolation and Transfection of Arabidopsis Leaf Mesophyll Protoplasts for Large-Scale Applications.

Arabidopsis leaf mesophyll protoplasts constitute an important and versatile tool for conducting cell-based experiments to analyze the functions of distinct signaling pathways and cellular machineries using proteomic, biochemical, cellular, genetic, and genomic approaches. Thus, the methods for protoplast isolation and transfection have been gradually improved to achieve efficient expression of genes of interest. Although many well-established protocols have been extensively tested, their successful application is sometimes limited to researchers with a high degree of skill and experience in protoplasts handling. Here we present a detailed method for the isolation and transfection of Arabidopsis mesophyll protoplasts, in which many of the time-consuming and critical steps present in the current protocols have been simplified. The method described is fast, simple, and leads to high yields of competent protoplasts allowing large-scale applications.

Metabolic Engineering of the Mevalonate and Non-Mevalonate Pathways in Tomato

In higher plants isoprenoids have many essential roles e.g. in membrane structure (phytosterols), redox chemistry (plastoquinone) and as antioxidants (carotenoids). Despite their functional and chemical diversity all isoprenoids are biosynthetically related from a common precursor, isopentenyl pyrophosphate (IPP). In higher plants two pathways exist for the formation of IPP, the mevalonate pathway where IPP is formed from acetyl-CoA via mevalonic acid (MVA) and a second, recently proven, pathway where IPP is formed from D-glyceraldehyde-3-phosphate and pyruvate via 1deoxy-D-xylulose 5-phosphate (DXP) (Rohmer, 1999). In the cytosol/endoplasmic reticulum (ER) and mitochondria the mevalonate pathway is responsible for the synthesis of phytosterols and ubiquinone. 3-Hydroxy-3-methylglutaryl Co-enzyme A reductase (HMGR) is a key regulatory enzyme in the mevalonate pathway and catalyses the formation of MVA from 3-hydroxy-3-methylglutaryl Co-A (HMG-CoA). Plastid isoprenoids such as carotenoids and tocopherols are formed via the non-MVA pathway. The formation of DXP, catalysed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), has been shown to be a regulator of flux through this pathway to carotenoids (Lois et al., 2000).