Maximization of fructose esters synthesis by response surface methodology

Enzymatic synthesis of fructose fatty acid ester was performed in organic solvent media, using a purified lipase from Candida antartica B immobilized in acrylic resin. Response surface methodology with a central composite rotatable design based on five levels was implemented to optimize three experimental operating conditions (temperature, agitation and reaction time). A statistical significant cubic model was established. Temperature and reaction time were found to be the most significant parameters. The optimum operational conditions for maximizing the synthesis of fructose esters were 57.1°C, 100 rpm and 37.8 h. The model was validated in the identified optimal conditions to check its adequacy and accuracy, and an experimental esterification percentage of 88.4% (±0.3%) was obtained. These results showed that an improvement of the enzymatic synthesis of fructose esters was obtained under the optimized conditions.     

Yeast aconitase in two locations and two metabolic pathways: seeing small amounts is believing

The distribution of identical enzymatic activities between different subcellular compartments is a fundamental process of living cells. At present, the Saccharomyces cerevisiae aconitase enzyme has been detected only in mitochondria, where it functions in the tricarboxylic acid (TCA) cycle and is considered a mitochondrial matrix marker. We developed two strategies for physical and functional detection of aconitase in the yeast cytosol: 1) we fused the alpha peptide of the beta-galactosidase enzyme to aconitase and observed alpha complementation in the cytosol; and 2) we created an ACO1-URA3 hybrid gene, which allowed isolation of strains in which the hybrid protein is exclusively targeted to mitochondria. These strains display a specific phenotype consistent with glyoxylate shunt elimination. Together, our data indicate that yeast aconitase isoenzymes distribute between two distinct subcellular compartments and participate in two separate metabolic pathways; the glyoxylate shunt in the cytosol and the TCA cycle in mitochondria. We maintain that such dual distribution phenomena have a wider occurrence than recorded currently, the reason being that in certain cases there is a small fraction of one of the isoenzymes, in one of the locations, making its detection very difficult. We term this phenomenon of highly uneven isoenzyme distribution "eclipsed distribution."     

Evidence for abundant transcription of non-coding regions in the Saccharomyces cerevisiae genome

Background

The ChickRH6 whole chicken genome radiation hybrid (RH) panel recently produced has already been used to build radiation hybrid maps for several chromosomes, generating comparative maps with the human and mouse genomes and suggesting improvements to the chicken draft sequence assembly. Here we present the construction of a RH map of chicken chromosome 2. Markers from the genetic map were used for alignment to the existing GGA2 (Gallus gallus chromosome 2) linkage group and EST were used to provide valuable comparative mapping information. Finally, all markers from the RH map were localised on the chicken draft sequence assembly to check for eventual discordances.

Results

Eighty eight microsatellite markers, 10 genes and 219 EST were selected from the genetic map or on the basis of available comparative mapping information. Out of these 317 markers, 270 gave reliable amplifications on the radiation hybrid panel and 198 were effectively assigned to GGA2. The final RH map is 2794 cR₆₀₀₀ long and is composed of 86 framework markers distributed in 5 groups. Conservation of synteny was found between GGA2 and eight human chromosomes, with segments of conserved gene order of varying lengths.

Conclusion

We obtained a radiation hybrid map of chicken chromosome 2. Comparison to the human genome indicated that most of the 8 groups of conserved synteny studied underwent internal rearrangements. The alignment of our RH map to the first draft of the chicken genome sequence assembly revealed a good agreement between both sets of data, indicative of a low error rate.     

Alg14 recruits Alg13 to the cytoplasmic face of the endoplasmic reticulum to form a novel bipartite UDP-N-acetylglucosamine transferase required for the second step of N-linked glycosylation

N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc2-PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc2-PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor.     

Root-synthesized cytokinin in Arabidopsis is distributed in the shoot by the transpiration stream

To clarify how root-synthesized cytokinins (CKs) are transported to young shoot organs, CK distribution patterns were analysed in free-CK-responsive ARR5::GUS transformants of Arabidopsis thaliana (L.) Heynh. together with free plus bound CKs using specific CK monoclonal antibodies. Plants were subjected to two different growth conditions, completely protected from any air movement, or exposed to gentle wind 3 h before harvesting. In wind-protected plants the strongest ARR5::GUS expression was found in the root cap statocytes, spreading upwards in the vascular cylinder. This pattern in roots was congruent with that found by CK immunolocalization. Shoots of wind-protected plants displayed either no or only low ARR5::GUS expression in the stem vascular bundles, nodal ramifications, and the bases of flower buds; shoot vascular bundles showed patterns of acropetally decreasing staining and the apical parts of buds and leaves were free from ARR5::GUS expression. In wind-exposed plants ARR5::GUS expression was considerably increased in shoots, also in basal-to-apical decreasing gradients. Immunolabelled shoots showed differential staining, with the strongest label in the vascular bundles of stems, leaves, and buds. The fact of the apparent absence of free CK in the buds of wind-protected plants and the typical upward decreasing gradients of free and conjugated CKs suggest that the bulk of the CK is synthesized in the root cap, exported through the xylem and accumulates at sites of highest transpiration where cuticles do not yet exist or do not protect against water loss.     

Heparanase uptake is mediated by cell membrane heparan sulfate proteoglycans

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.     

Hetero-assembly between all-L- and all-D-amino acid transmembrane domains: forces involved and implication for inactivation of membrane proteins

Protein-protein interactions within the membrane, partially or fully mediated by transmembrane (TM) domains, are involved in many vital cellular processes. Since the unique feature of the membrane environment enables protein-protein assembly that otherwise is not energetically favored in solution, the structural restrictions involved in the assembly of soluble proteins are not necessarily valid for the assembly of TM domains. Here we used the N-terminal TM domain (Tar-1) of the Escherichia coli aspartate receptor as a model system for examining the stereospecificity of TM-TM interactions in vitro and in vivo in isolated systems, and in the context of the full receptor. For this propose, we synthesized Tar-1 all-l and all-d amino acid TM peptides, a mutant TM peptide and an unrelated TM peptide. The data revealed: (i) Tar-1 all-d specifically associated with Tar-1 all-l within a model lipid membrane, as determined by using fluorescence energy transfer experiments; (ii) Tar-1 all-l and all-d, but not the control peptides, demonstrated a dose-dependant dominant negative effect on the Tar-1 TM homodimerization in the bacterial ToxR assembly system, suggesting a wild-type-like interaction; and most interestingly, (iii) both Tar-1 all-l and all-d showed a remarkable ability to inhibit the chemotaxis response of the full-length receptor, in vivo. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blotting confirmed that ToxR Tar-1 chimera protein levels are not affected by the presence of the exogenous peptides. These findings present the first evidence that an all-d TM domain peptide acts in vivo similarly to its parental all-l peptide and suggest that the dimerization of the TM domains is mainly mediated by side-chain interactions, rather than geometrically fitted conformations. In addition, the study provides a new approach for modifying the function of membrane proteins by proteolysis-free peptides.     

Genetic evidence for functional interaction of the Escherichia coli signal recognition particle receptor with acidic lipids in vivo

The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding α-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.