RESUMEN
The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-ß-d-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.
Asunto(s)
Escherichia coli/metabolismo , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Fosfolípidos/metabolismo , Fotoperiodo , Rodopsinas Microbianas/metabolismo , Detergentes/química , Detergentes/metabolismo , Cinética , Membrana Dobles de Lípidos/metabolismo , Liposomas , Espectroscopía de Resonancia Magnética , Lípidos de la Membrana/metabolismo , Fotólisis , Rodopsinas Microbianas/efectos de la radiaciónRESUMEN
The Cpx stress response system is induced by various environmental and cellular stimuli. It is also activated in Escherichia coli strains lacking the major phospholipid, phosphatidylethanolamine (PE). However, it is not known whether CpxA directly senses changes in the lipid bilayer or the presence of misfolded proteins due to the lack of PE in their membranes. To address this question, we used an in vitro reconstitution system and vesicles with different lipid compositions to track modulations in the activity of CpxA in different lipid bilayers. Moreover, the Cpx response was validated in vivo by monitoring expression of a PcpxP-gfp reporter in lipid-engineered strains of E. coli. Our combined data indicate that CpxA responds specifically to different lipid compositions.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Membrana Dobles de Lípidos/química , Modelos Moleculares , Fosfatidiletanolaminas/química , Proteínas Quinasas/química , Procesamiento Proteico-Postraduccional , Transducción de Señal , Acholeplasma laidlawii/enzimología , Acholeplasma laidlawii/metabolismo , Arabidopsis/enzimología , Arabidopsis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cardiolipinas/química , Cardiolipinas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Reporteros , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceroles/química , Fosfatidilgliceroles/metabolismo , Fosforilación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Propiedades de SuperficieRESUMEN
The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.
Asunto(s)
Escherichia coli/metabolismo , Ácidos Grasos/metabolismo , Glucosiltransferasas/metabolismo , Acholeplasma laidlawii/enzimología , Acholeplasma laidlawii/genética , Acholeplasma laidlawii/metabolismo , Membrana Celular/enzimología , Membrana Celular/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Metiltransferasas/metabolismo , Modelos Moleculares , Fosfatidiletanolaminas/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by (31)P NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a "feed-forward" stimulation of lipid synthesis in E. coli. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.
Asunto(s)
Acholeplasma laidlawii/enzimología , Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , Glucosiltransferasas/metabolismo , Lípidos de la Membrana/metabolismo , Fosfolípidos/biosíntesis , Acetatos/metabolismo , Acholeplasma laidlawii/genética , Aniones/química , Aniones/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Unión Competitiva , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/genética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Espectroscopía de Resonancia Magnética , Lípidos de la Membrana/química , Modelos Moleculares , Análisis Multivariante , Mutación , Fosfolípidos/química , Unión Proteica , Estructura Terciaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , Transformación GenéticaRESUMEN
Chloroplast membranes contain a substantial excess of the nonbilayer-prone monogalactosyldiacylglycerol (GalDAG) over the biosynthetically consecutive, bilayer-forming digalactosyldiacylglycerol (GalGalDAG), yielding a high membrane curvature stress. During phosphate shortage, plants replace phospholipids with GalGalDAG to rescue phosphate while maintaining membrane homeostasis. Here we investigate how the activity of the corresponding glycosyltransferase (GT) in Arabidopsis thaliana (atDGD2) depends on local bilayer properties by analyzing structural and activity features of recombinant protein. Fold recognition and sequence analyses revealed a two-domain GT-B monotopic structure, present in other plant and bacterial glycolipid GTs, such as the major chloroplast GalGalDAG GT atDGD1. Modeling led to the identification of catalytically important residues in the active site of atDGD2 by site-directed mutagenesis. The DGD synthases share unique bilayer interface segments containing conserved tryptophan residues that are crucial for activity and for membrane association. More detailed localization studies and liposome binding analyses indicate differentiated anchor and substrate-binding functions for these separated enzyme interface regions. Anionic phospholipids, but not curvature-increasing nonbilayer lipids, strongly stimulate enzyme activity. From our studies, we propose a model for bilayer "control" of enzyme activity, where two tryptophan segments act as interface anchor points to keep the substrate region close to the membrane surface. Binding of the acceptor substrate is achieved by interaction of positive charges in a surface cluster of lysines, arginines, and histidines with the surrounding anionic phospholipids. The diminishing phospholipid fraction during phosphate shortage stress will then set the new GalGalDAG/phospholipid balance by decreasing stimulation of atDGD2.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Membrana Celular/enzimología , Galactosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Estrés Fisiológico/fisiología , Triptófano/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/genética , Galactosiltransferasas/genética , Proteínas de la Membrana/genética , Modelos Biológicos , Análisis de Secuencia de Proteína , Triptófano/genéticaRESUMEN
Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227-245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130-148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227-245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Glicosiltransferasas/metabolismo , Metabolismo de los Lípidos , Fosfatos/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Dicroismo Circular , Glicosiltransferasas/química , Datos de Secuencia Molecular , Análisis Multivariante , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Espectrometría de FluorescenciaRESUMEN
Cyanobacteria are unique eubacteria with an organized subcellular compartmentalization of highly differentiated internal thylakoid membranes (TM), in addition to the outer and plasma membranes (PM). This leads to a complicated system for transport and sorting of proteins into the different membranes and compartments. By shotgun and gel-based proteomics of plasma and thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803, a large number of membrane proteins were identified. Proteins localized uniquely in each membrane were used as a platform describing a model for cellular membrane organization and protein intermembrane sorting and were analyzed by multivariate sequence analyses to trace potential differences in sequence properties important for insertion and sorting to the correct membrane. Sequence traits in the C-terminal region, but not in the N-terminal nor in any individual transmembrane segments, were discriminatory between the TM and PM classes. The results are consistent with a contact zone between plasma and thylakoid membranes, which may contain short-lived "hemifusion" protein traffic connection assemblies. Insertion of both integral and peripheral membrane proteins is suggested to occur through common translocons in these subdomains, followed by a potential translation arrest and structure-based sorting into the correct membrane compartment.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Proteómica , Synechocystis/metabolismo , Proteínas Bacterianas/química , Western Blotting , Electroforesis en Gel de Poliacrilamida , Proteínas de la Membrana/química , Análisis Multivariante , Espectrometría de Masas en TándemRESUMEN
As in other eukaryotes, plant plasma membranes contain sphingolipids, phospholipids, and free sterols. In addition, plant plasma membranes also contain sterol derivatives and usually <5 mol% of a galactolipid, digalactosyldiacylglycerol (DGDG). We earlier reported that compared to fully fertilized oats (Avena sativa), oats cultivated without phosphate replaced up to 70 mol% of the root plasma membrane phospholipids with DGDG. Here, we investigated the implications of a high DGDG content on membrane properties. The phospholipid-to-DGDG replacement almost exclusively occurred in the cytosolic leaflet, where DGDG constituted up to one-third of the lipids. In the apoplastic (exoplasmic) leaflet, as well as in rafts, phospholipids were not replaced by DGDG, but by acylated sterol glycosides. Liposome studies revealed that the chain ordering in free sterol/phospholipid mixtures clearly decreased when >5 mol% DGDG was included. As both the apoplastic plasma membrane leaflet (probably the major water permeability barrier) and rafts both contain only trace amounts of DGDG, we conclude that this lipid class is not compatible with membrane functions requiring a high degree of lipid order. By not replacing phospholipids site specifically with DGDG, negative functional effects of this lipid in the plasma membrane are avoided.-Tjellström, H., Hellgren, L. I., Wieslander, A., Sandelius, A. S. Lipid asymmetry in plant plasma membranes: phosphate deficiency-induced phospholipid replacement is restricted to the cytosolic leaflet.
Asunto(s)
Avena/metabolismo , Galactolípidos/metabolismo , Microdominios de Membrana/metabolismo , Fosfatos/deficiencia , Fosfolípidos/metabolismo , Esteroles/metabolismo , Acetilación , Glicósidos/metabolismoRESUMEN
The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of > or = 60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter approximately 100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.
Asunto(s)
Membrana Celular/metabolismo , Escherichia coli/metabolismo , Glicosiltransferasas/química , Membranas Intracelulares/metabolismo , Lípidos/química , Lípidos de la Membrana/química , Proteínas Bacterianas/química , Estructuras Celulares/metabolismo , Centrifugación por Gradiente de Densidad/métodos , Microscopía por Crioelectrón/métodos , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/metabolismo , Modelos Moleculares , Conformación Molecular , Fosfolípidos/químicaRESUMEN
We have used multivariate statistics and z-scales to represent peptide sequences in a PLS-QSAR model of previously studied binding affinities [Kofler,M., Motzny,K. and Freund,C. (2005b) Mol. Cell. Proteomics, 4, 1797-1811.] of two GYF domains to an array of immobilized synthetic peptides. As a result, we established structural determinants of the binding specificities of the two proteins. Our model was used to define new sets of yeast proteins potentially interacting with Syh1 (YPL105C) and Smy2 (YBR172C). These sets were subsequently examined for co-occurrence of Gene Ontology terms, leading to suggest a group of likely interacting proteins with a common function in mRNA catabolism. Finally, subcellular localization of a GFP-fused Syh1 and Smy2 reinforced the possibility that these proteins reside in cytoplasmic sites of mRNA degradation, thereby providing experimental confirmation to the predictions from the model.
Asunto(s)
Saccharomyces cerevisiae/metabolismo , Algoritmos , Citoplasma/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Análisis de los Mínimos Cuadrados , Modelos Teóricos , Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Programas Informáticos , Relación Estructura-ActividadRESUMEN
One of the major lipids in the membranes of Borrelia burgdorferi is monogalactosyl diacylglycerol (MGalDAG), a glycolipid recently shown to carry antigenic potency. Herein, it is shown that the gene mgs (TIGR designation bb0454) of B. burgdorferi encodes for the protein bbMGS that, when expressed in Escherichia coli, catalyzes the glycosylation of 1,2-diacylglycerol with specificity for the donor substrate UDP-Gal yielding MGalDAG. Related lipid enzymes were found in many Gram-positive bacteria. The presence of this galactosyltransferase activity and synthesis of a cholesteryl galactoside by another enzyme were verified in B. burgdorferi cell extract. Besides MGalDAG, phosphatidylcholine, phosphatidylglycerol, and cholesterol were also found as major lipids in the cell envelope. The high isoelectric point of bbMGS and clustered basic residues in its amino acid sequence suggest that the enzyme interacts with acidic lipids in the plasma membrane, in agreement with strong enzymatic activation of bbMGS by phosphatidylglycerol. The membrane packing and immunological properties of MGalDAG are likely to be of great importance in vivo.
Asunto(s)
Proteínas Bacterianas/metabolismo , Borrelia burgdorferi/enzimología , Diglicéridos/biosíntesis , Galactolípidos/metabolismo , Galactosiltransferasas/metabolismo , Metabolismo de los Lípidos , Lípidos de la Membrana/biosíntesis , Proteínas Bacterianas/genética , Borrelia burgdorferi/genética , Membrana Celular/química , Colesterol/análisis , Colesterol/aislamiento & purificación , Clonación Molecular , Diglicéridos/metabolismo , Activadores de Enzimas/análisis , Activadores de Enzimas/aislamiento & purificación , Activadores de Enzimas/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosiltransferasas/genética , Genes Bacterianos , Punto Isoeléctrico , Fosfatidilcolinas/análisis , Fosfatidilcolinas/aislamiento & purificación , Fosfatidilgliceroles/análisis , Fosfatidilgliceroles/aislamiento & purificación , Fosfatidilgliceroles/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de Proteína , Transcripción Genética , Uridina Difosfato Galactosa/metabolismoRESUMEN
WaaG is a glycosyltransferase that is involved in the biosynthesis of lipopolysaccharide in Gram-negative bacteria. Inhibitors of WaaG are highly sought after as they could be used to inhibit the biosynthesis of the core region of lipopolysaccharide, which would improve the uptake of antibiotics. Herein, we establish an activity assay for WaaG using (14)C-labeled UDP-glucose and LPS purified from a ∆waaG strain of Escherichia coli. We noted that addition of the lipids phosphatidylglycerol (PG) and cardiolipin (CL), as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) increased activity. We then use the assay to determine if three molecular scaffolds, which bind to WaaG, could inhibit its activity in vitro. We show that 4-(2-amino-1,3-thiazol-4-yl)phenol inhibits WaaG (IC50 1.0 mM), but that the other scaffolds do not. This study represents an important step towards an inhibitor of WaaG by fragment-based lead discovery.
RESUMEN
The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Modelos Estructurales , Simportadores de Protón-Fosfato/química , Simportadores de Protón-Fosfato/aislamiento & purificación , Simportadores de Protón-Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Western Blotting , Cromatografía de Afinidad , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Proteínas Fúngicas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Simportadores de Protón-Fosfato/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Marcadores de SpinRESUMEN
Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide (TP) directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide (dTP). Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis (MVDA) with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed. dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an 'avoidance signal' for the chloroplast import.
Asunto(s)
Arabidopsis/citología , Cloroplastos/metabolismo , Mitocondrias/metabolismo , Péptidos/metabolismo , Arabidopsis/metabolismo , Biología Computacional , Mutagénesis , Péptidos/química , Péptidos/genética , Señales de Clasificación de Proteína , Transporte de ProteínasRESUMEN
Synthesis of monogalactosyldiacylglycerol (GalDAG) and digalactosyldiacylglycerol (GalGalDAG), the major membrane lipids in cyanobacteria, begins with production of the intermediate precursor monoglucosyldiacylglycerol (GlcDAG), by monoglucosyldiacylglycerol synthase (MGS). In Synechocystis sp. PCC6803 (Synechocystis) this activity is catalyzed by an integral membrane protein, Sll1377 or MgdA. In silico sequence analysis revealed that cyanobacterial homologues of MgdA are highly conserved and comprise a distinct group of lipid glycosyltransferases. Global regulation of lipid synthesis in Synechocystis and, more specifically, the influence of the lipid environment on MgdA activity have not yet been fully elucidated. Therefore, we purified membrane subfractions from this organism and assayed MGS activity in vitro, with and without different lipids and other potential effectors. Sulfoquinovosyldiacylglycerol (SQDAG) potently stimulates MgdA activity, in contrast to other enzymes of a similar nature, which are activated by phosphatidylglycerol instead. Moreover, the final products of galactolipid synthesis, GalDAG and GalGalDAG, inhibited this activity. Western blotting revealed the presence of MgdA both in plasma and thylakoid membranes, with a high specific level of the MgdA protein in the plasma membrane but highest MGS activity in the thylakoid membrane. This discrepancy in the subcellular localization of enzyme activity and protein may indicate the presence of either an unknown regulator and/or an as yet unidentified MGS-type enzyme. Furthermore, the stimulation of MgdA activity by SQDAG observed here provides a new insight into regulation of the biogenesis of both sulfolipids and galactolipids in cyanobacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Glucosiltransferasas/metabolismo , Lípidos/química , Synechocystis/enzimología , Secuencia de Aminoácidos , Vías Biosintéticas/efectos de los fármacos , Western Blotting , Isótopos de Carbono , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cromatografía en Capa Delgada , Secuencia Conservada , Activadores de Enzimas/farmacología , Glucosiltransferasas/química , Glucolípidos/farmacología , Lípidos/biosíntesis , Micelas , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , Synechocystis/efectos de los fármacosRESUMEN
Intracellular vesicles are abundant in eukaryotic cells but absent in the Gram-negative bacterium Escherichia coli. However, strong overexpression of a monotopic glycolipid-synthesizing enzyme, monoglucosyldiacylglycerol synthase from Acholeplasma laidlawii (alMGS), leads to massive formation of vesicles in the cytoplasm of E. coli. More importantly, alMGS provides a model system for the regulation of membrane properties by membrane-bound enzymes, which is critical for maintaining cellular integrity. Both phenomena depend on how alMGS binds to cell membranes, which is not well understood. Here, we carry out a comprehensive investigation of the membrane binding of alMGS by combining bioinformatics methods with extensive biochemical studies, structural modeling and molecular dynamics simulations. We find that alMGS binds to the membrane in a fairly upright manner, mainly by residues in the N-terminal domain, and in a way that induces local enrichment of anionic lipids and a local curvature deformation. Furthermore, several alMGS variants resulting from substitution of residues in the membrane anchoring segment are still able to generate vesicles, regardless of enzymatic activity. These results clarify earlier theories about the driving forces for vesicle formation, and shed new light on the membrane binding properties and enzymatic mechanism of alMGS and related monotopic GT-B fold glycosyltransferases.
Asunto(s)
Proteínas Bacterianas/fisiología , Vesículas Citoplasmáticas/enzimología , Glicosiltransferasas/fisiología , Acholeplasma laidlawii/enzimología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Membrana Celular/enzimología , Escherichia coli/enzimología , Escherichia coli/ultraestructura , Glicosiltransferasas/química , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The Mycobacterium tuberculosis acid-induced operon MymA encodes the fatty acyl-CoA synthetase FadD13 and is essential for virulence and intracellular growth of the pathogen. Fatty acyl-CoA synthetases activate lipids before entering into the metabolic pathways and are also involved in transmembrane lipid transport. Unlike soluble fatty acyl-CoA synthetases, but like the mammalian integral-membrane very-long-chain acyl-CoA synthetases, FadD13 accepts lipid substrates up to the maximum length tested (C(26)). Here, we show that FadD13 is a peripheral membrane protein. The structure and mutational studies reveal an arginine- and aromatic-rich surface patch as the site for membrane interaction. The protein accommodates a hydrophobic tunnel that extends from the active site toward the positive patch and is sealed by an arginine-rich lid-loop at the protein surface. Based on this and previous data, we propose a structural basis for accommodation of lipid substrates longer than the enzyme and transmembrane lipid transport by vectorial CoA-esterification.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Coenzima A Ligasas/química , Mycobacterium tuberculosis/enzimología , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Dominio Catalítico , Coenzima A Ligasas/aislamiento & purificación , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Propiedades de SuperficieRESUMEN
Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco- and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayer-prone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayer-prone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyl-diacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.
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Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Metabolismo de los Lípidos , Transporte Biológico , Permeabilidad de la Membrana Celular , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Metabolismo de los Lípidos/genética , Viabilidad Microbiana/efectos de los fármacos , Mutación/genética , Presión Osmótica , Ingeniería de Proteínas , Tolerancia a la SalRESUMEN
Cyanobacteria have a cell envelope consisting of a plasma membrane, a periplasmic space with a peptidoglycan layer, and an outer membrane. A third, separate membrane system, the intracellular thylakoid membranes, is the site for both photosynthesis and respiration. All membranes and luminal spaces have unique protein compositions, which impose an intriguing mechanism for protein sorting of extracytoplasmic proteins due to single sets of translocation protein genes. It is shown here by multivariate sequence analyses of many experimentally identified proteins in Synechocystis, that proteins routed for the different extracytosolic compartments have correspondingly different physicochemical properties in their signal peptide and mature N-terminal segments. The full-length mature sequences contain less significant information. From these multivariate, N-terminal property-profile models for proteins with single experimental localization, proteins with ambiguous localization could, to a large extent, be predicted to a defined compartment. The sequence properties involve amino acids varying especially in volume and polarizability and at certain positions in the sequence segments, in a manner typical for the various compartment classes. Potential means of the cell to recognize the property features are discussed, involving the translocation channels and two Type I signal peptidases with different cellular localization, and charge features at their membrane interfaces.
Asunto(s)
Proteínas Bacterianas/química , Proteoma/análisis , Análisis de Secuencia de Proteína , Synechocystis/química , Secuencia de Aminoácidos , Proteínas Bacterianas/análisis , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Proteínas de Transporte de Membrana/análisis , Proteínas de Transporte de Membrana/química , Datos de Secuencia Molecular , Análisis Multivariante , Transporte de Proteínas , Proteómica , Serina Endopeptidasas/análisis , Serina Endopeptidasas/químicaRESUMEN
The human pathogen Mycoplasma pneumoniae has a very small genome but with many yet not identified gene functions, e.g. for membrane lipid biosynthesis. Extensive radioactive labelling in vivo and enzyme assays in vitro revealed a substantial capacity for membrane glycolipid biosynthesis, yielding three glycolipids, five phosphoglycolipids, in addition to six phospholipids. Most glycolipids were synthesized in a cell protein/lipid-detergent extract in vitro; galactose was incorporated into all species, whereas glucose only into a few. One (MPN483) of the three predicted glycosyltransferases (GTs; all essential) was both processive and promiscuous, synthesizing most of the identified glycolipids. These enzymes are of a GT-A fold, similar to an established structure, and belong to CAZy GT-family 2. The cloned MPN483 could use both diacylglycerol (DAG) and human ceramide acceptor substrates, and in particular UDP-galactose but also UDP-glucose as donors, making mono-, di- and trihexose variants. MPN483 output and processitivity was strongly influenced by the local lipid environment of anionic lipids. The structure of a major beta1,6GlcbetaGalDAG species was determined by NMR spectroscopy. This, as well as other purified M. pneumoniae glycolipid species, is important antigens in early infections, as revealed from ELISA screens with patient IgM sera, highlighting new aspects of glycolipid function.