RESUMEN
Conditions for simple derivatization of reducing carbohydrates via adipic acid dihydrazide microwave-assisted condensation are described. We demonstrate with a diverse set of oligo- and polysaccharides how to improve a restrictive and labor intensive conventional conjugation protocol by using microwave-assisted chemistry. We show that 5 min of microwave heating in basic or acidic conditions are adequate to generate, in increased yields, intact and functional glycosylhydrazides, whereas hours to days and acidic conditions are generally required under conventional methods.
Asunto(s)
Carbohidratos/síntesis química , Microondas , Secuencia de Carbohidratos , Carbohidratos/química , Resonancia por Plasmón de SuperficieRESUMEN
The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [125I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53-56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp56 into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Fúngicas/metabolismo , Phytophthora/metabolismo , Secuencias de Aminoácidos , Membrana Celular/metabolismo , Pared Celular/metabolismo , Proteínas de la Membrana/metabolismo , Oligopéptidos/metabolismoRESUMEN
A cDNA clone, AtELP1 (Arabidopsis thaliana EGF receptor-like protein) was isolated from an Arabidopsis cDNA library with an oligonucleotide probe corresponding to a highly conserved region of animal beta-integrins. The cloning of this cDNA was previously reported and it has been proposed that AtELP might be a receptor involved in intracellular trafficking. In the present work, using two specific independent sets of anti-peptide antibodies, we show that AtELP1 is mainly located in the plasma membrane, supporting another function for this protein. Structural studies, using methods for secondary structure prediction, indicated the presence of cysteine-rich domains specific to beta-integrins. Database searches revealed that AtELP1 is a member of a multigenic family composed of at least six members in A. thaliana. Northern blot analysis of AtELP1, 2b and 3 was performed on mRNA extracted from cells cultured in normal and stressed conditions, and from several organs and plants submitted to biotic or abiotic stresses. All the genes are expressed at different levels in the same conditions, but preferentially in roots, fruits and leaves in response to water deficit.
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Arabidopsis/genética , Receptores ErbB/genética , Integrina beta1/genética , Secuencia de Aminoácidos , Northern Blotting , Western Blotting , Clonación Molecular , ADN Complementario/química , Regulación de la Expresión Génica de las Plantas , Integrina beta1/biosíntesis , Integrina beta1/química , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/química , Análisis de SecuenciaRESUMEN
An Arabidopsis cDNA clone that defines a new class of plant serine/threonine receptor kinases was found to be a member of a family of four clustered genes (lecRK-a1-a4) which have been cloned, sequenced and mapped on chromosome 3. This family belongs to a large superfamily encoding putative receptors with an extracellular domain homologous to legume lectins and appears to be conserved at least among dicots. In the Columbia ecotype only the lecRK-a1 and perhaps the lecRK-a3 gene is functional, since lecRK-a2 is disrupted by a Ty-copia retroelement and lecRK-a4 contains a frameshift mutation. Structural analysis of the lecRK-al and lecRK-a3 deduced amino-acid sequences suggests that the lectin domain is unlikely to be involved in binding monosaccharides but could interact with complex glycans and/or with hydrophobic ligands. Immunodetection of lecRK gene products in plasma membranes purified by free-flow electrophoresis showed that the lecRK-a proteins are probably highly glycosylated integral plasma membrane components.
Asunto(s)
Arabidopsis/genética , Proteínas de Plantas/genética , Receptores Mitogénicos/genética , Secuencia de Aminoácidos , Sitios de Unión , Mapeo Cromosómico , Evolución Molecular , Fabaceae/genética , Genes de Plantas , Lectinas/química , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Lectinas de Plantas , Plantas Medicinales , Conformación Proteica , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Rhizobial lipo-chitooligosaccharides (LCOs) are signaling molecules involved in host-range recognition for the establishment of the symbiosis with leguminous plants. The major LCO of Rhizobium meliloti, the symbiont of Medicago plants contains four or five N-acetylglucosamines, O-acetylated and N-acylated with a C16:2 fatty acid on the terminal nonreducing sugar and O-sulfated on the reducing sugar. In this paper, the ligand specificity of a high-affinity binding site (Nod factor binding site 2 or NFBS2), enriched in a plasma membrane-enriched fraction of Medicago cell suspension cultures, is reported. By using chemically synthesized LCOs, the role of structural elements, important for symbiotic activities, as recognition motifs for NFBS2 was determined. The results show that the substitutions on the nonreducing sugar of the LCOs (the O-acetate group, the fatty acid, and the hydroxyl group on the C4 of the sugar) are determinants for high-affinity binding to NFBS2. In contrast, the sulfate group, which is necessary for all biological activities on Medicago, is not discriminated by NFBS2. However, the reducing sugar of the LCO seems to interact with NFBS2, because ligand binding is affected by the reduction of the free anomeric carbon and depends on the number of N-acetyl glucosamine residues. These results suggest that the recognition of the LCOs by NFBS2 is mediated by structural elements in both the lipid and oligosaccharidic moities, but not by the sulfate group.
RESUMEN
The review focuses on the multiple separating regimes that offers the free flow electrophoresis technique: free flow zone electrophoresis, isoelectric focusing, isotachophoresis, free flow step electrophoresis. Also, the feasibility to apply either interval or continuous flow electrophoresis is evaluated. The free flow zone electrophoresis regime is generally selected for the separation of cells, organelles and membranes while the other regimes find their largest fields of applications in the purification of proteins and peptides. The latter regimes present the highest resolution efficiency. Therefore, a large part of this review is devoted to the applicabilities of these different regimes to the purification of organelles and membrane vesicles at the preparative scale. Recent developments, both in instrumentation and procedures, are described. The major achievements in plant membrane fractionation obtained with free flow electrophoresis are outlined. The related procedures are both analytical and preparative: they separate tonoplast and plasma membrane simultaneously from the same homogenate, they discriminate for one type of membrane vesicles of opposite orientation, and process large quantities of membrane material by reason of the continuous flow mode. Recent advances using electromigration techniques that permit confirmation of the dynamic state of membranes, characterisation of complex membrane-dependent functions and discovery of new membrane-localised activities are presented.
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Membrana Celular , Electroforesis/métodos , Células VegetalesRESUMEN
We constructed a high-efficiency expression library from Arabidopsis cDNA clones by introducing a poly (dC) stretch at the 5' end of the clones. This library enables the synthesis of proteins from all the cDNA clones present. We have screened the high-efficiency expression library with antibodies raised against total proteins from Arabidopsis plasmalemma and tonoplast. With the positive clones, we have constructed two cDNA ordered libraries enriched in genes encoding plasmalemma (522 clones) and tonoplast proteins (594 clones). Partial sequencing of both libraries shows that a high proportion (47%) of the clones encoded putative membrane proteins, or membrane-associated proteins. When sequenced, 55% of the cDNAs were new EST sequences for Arabidopsis, 26% were similar to genes present in other plants or organisms, and 29% were not referenced in any databank. Immunoscreening of the two cDNA ordered libraries with antibodies raised against proteins from Arabidopsis cells submitted to osmotic stress allows the selection of genes over- and under-expressed in stress conditions.
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Arabidopsis/genética , ADN Complementario , Biblioteca de Genes , Proteínas de Plantas/genética , Arabidopsis/metabolismo , Secuencia de Bases , Membrana Celular/metabolismo , Orgánulos/metabolismo , Proteínas de Plantas/biosíntesis , Poli CRESUMEN
The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.
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Arabidopsis/metabolismo , Oligopéptidos/metabolismo , Proteínas de Plantas/metabolismo , Arabidopsis/ultraestructura , Sitios de Unión , Adhesión Celular , Membrana Celular/metabolismo , Pared Celular/metabolismo , Cinética , CebollasRESUMEN
Endo-1,4-beta-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels. However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose-hemicellulose network in the expanding cell wall.
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Arabidopsis/enzimología , Membrana Celular/enzimología , Pared Celular/metabolismo , Celulasa/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Arabidopsis/ultraestructura , División Celular , Pared Celular/ultraestructura , Clonación Molecular , Secuencia Conservada , Evolución Molecular , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Hipocótilo , Datos de Secuencia Molecular , Mutación , Polisacáridos/metabolismo , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Highly purified tonoplast and plasma membrane vesicles were isolated from microsomes of Arabidopsis thaliana by preparative free-flow electrophoresis. The most electronegative fractions were identified as tonoplast using nitrate-inhibited Mg2+-ATPase as enzyme marker. The least electronegative fractions were identified as plasma membrane using glucan-synthase II, UDPG: sterol-glucosyl-transferase, and vanadate-inhibited Mg2+-ATPase as enzyme markers. Other membrane markers, latent inosine-5'-diphosphatase (Golgi), NADPH-cytochrome-c reductase (endoplasmic reticulum) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane. Immunoblot analysis of membrane fractions by antibodies directed against tonoplast and plasma membrane proteins confirmed the nature and the purity of the isolated membranes. The cytoskeletal protein actin, which was also identified by immunoblotting, was found to be specifically attached to the plasma membrane vesicles. The structural and functional integrity of the isolated membranes from Arabidopsis thaliana is discussed in the light of results obtained for the location of receptors and enzymes, or for the determination of ligand binding activity.
Asunto(s)
Fraccionamiento Celular/métodos , Electroforesis/métodos , Proteínas de la Membrana/análisis , Reacciones Antígeno-Anticuerpo , Arabidopsis , Biomarcadores/análisis , Membrana Celular/química , Membrana Celular/enzimología , Membrana Celular/inmunología , Cloroplastos/química , Cloroplastos/inmunología , Membranas Intracelulares/química , Membranas Intracelulares/enzimología , Proteínas de la Membrana/inmunología , Proteínas de Plantas/análisis , Fracciones Subcelulares/química , Fracciones Subcelulares/enzimologíaRESUMEN
A cDNA clone At.MAMI (Arabidopsis thaliana membrane-associated mannitol-induced) was isolated from an Arabidopsis cDNA expression library by immunoselection. The cDNA was full-length (1.18 kb) with an open reading frame of 798 nucleotides encoding a 265 amino acid protein. The sequence of At.MAMI did not show any significant identity with other genes, as well as the deduced amino acid sequence with other proteins. However, prediction methods for the secondary structure of MAMI-30, together with homologous domains revealed some identity with VAP-33, a protein involved in membrane trafficking in neuronal tissues. In contrast to VAP-33, MAMI-30 did not exhibit a transmembrane domain, but positively charged loop regions could be involved in membrane anchoring. Indeed, MAMI-30 was immunodetected in purified plasma membrane from Arabidopsis cells. The gene was responsive to low turgor in Arabidopsis and its expression regulated developmentally. In addition, reduction of turgor caused a higher accumulation of mRNAs.
Asunto(s)
Arabidopsis/química , Proteínas de Plantas/química , Secuencia de Aminoácidos , Anticuerpos , Arabidopsis/genética , Secuencia de Bases , Membrana Celular/química , Células Cultivadas , ADN Complementario/inmunología , ADN Complementario/aislamiento & purificación , Expresión Génica , Datos de Secuencia Molecular , Presión Osmótica , Proteínas de Plantas/genética , Estructura Secundaria de Proteína , Receptores de Vitronectina/inmunologíaRESUMEN
Two cell lines of Eucalyptus gunnii have been shown to keep their differential frost tolerance at the cellular level after long-term culture. They have been used to investigate the fluidity of specific cell membranes in relation with frost tolerance. Protoplasts and isolated vacuoles were obtained from both cell lines. In addition, purified plasma membrane and tonoplast (the vacuolar membrane) were separated from a crude microsomal fraction through free-flow electrophoresis. The lateral and rotational mobilities of lipids in these different membranes were studied by two biophysical techniques: fluorescence recovery after photobleaching (FRAP) and fluorescence polarization. After labeling the vacuoles isolated from the frost-sensitive cells with 1-oleoyl-2-(7-nitro-2,1,3-benz-oxadiazol-4-yl)aminocaproyl phosphatidylcholine, a single mobile component was observed with a diffusion coefficient of 2.4 x 10(-9) cm(2) s(-1) and a mobile fraction close to 100% at a temperature of 23 degrees C. When using isolated vacuoles from the frost tolerant line, a higher lateral diffusion of tonoplast lipids was found with a diffusion coefficient of 3.2 x 10(-9) cm(2) s(-1), still with a mobile fraction close to 100%. No convincing data were obtained when performing fluorescence recovery after photobleaching experiments on protoplasts. Fluorescence polarization experiments confirmed the differential behavior of the two cell lines for tonoplast and also for plasma membrane. In addition, they showed that intrinsically tonoplast exhibited a higher fluidity than plasma membrane. Our results provide the first information on the fluidity of tonoplast and on the compared properties of two important plant membranes-tonoplast and plasma membrane-through the use of two complementary biophysical approaches. In addition, they suggest there is a correlation between membrane fluidity and cold tolerance. The potential interest of plant vacuole as a natural model system in membrane studies is emphasized.
RESUMEN
The involvement of a histidyl residue in the binding or translocation step was investigated in the malate carrier at the tonoplast of Catharanthus roseus cells. The transport rate was strongly stimulated when the pH of the incubation medium was decreased from pH 7.0 to 5.0. The histidine-specific reagent diethylpyrocarbonate (DEPC) efficiently inhibited the activity of the malate carrier. Inhibition developed rapidly and was completed after 5 min at a concentration of 2 mM DEPC. The original substrate, malate, partially protected the carrier from inactivation by DEPC. Other organic acids (citrate, quinate) which are known to affect the malate transport of isolated vacuoles or tonoplast vesicles also showed protective properties. Inhibition of malate transport on tonoplast vesicles can also be achieved by photooxidation in the presence of the dye Rose Bengal. Malate also proved to protect against inactivation. The results strongly support the notion that a histidyl residue(s) is involved either in the binding or translocation of malate and that the protonation of the histidyl residue is essential to provide a high rate of malate transport.
Asunto(s)
Proteínas Portadoras/química , Histidina/análisis , Malatos/análisis , Células Vegetales , Vacuolas/química , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Proteínas Portadoras/fisiología , Células Cultivadas , Dietil Pirocarbonato/farmacología , Concentración de Iones de Hidrógeno , Malatos/farmacocinética , Fenómenos Fisiológicos de las Plantas , Plantas/metabolismo , Rosa Bengala/farmacología , Vacuolas/metabolismoRESUMEN
Highly purified tonoplast and plasmamembrane vesicles were isolated from microsomes of Catharanthus roseus (L.) G. Don. by preparative free-flow electrophoresis. The relative amounts of tonoplast and plasma-membrane vesicles in the total microsomes varied with the pH of the grinding medium. The most electronegative fractions were identified as tonoplast using nitrate-inhibited, azide-resistant Mg(2+)-ATPase and pyrophosphatase activities as enzyme markers. The least electronegative fractions were identified as plasma membrane using glucan-synthase-II and UDPG:sterolglucosyl-transferase activities as enzyme markers. Other membrane markers, latent inosine-5'-diphosphatase (Golgi), NADPH-cytochrome-c reductase (ER) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane and did not contaminate either the tonoplast or the plasma-membrane fractions. In the course of searching for a reliable marker for tonoplast, the pyrophosphatase activity was found to be essentially associated with the tonoplast fractions purified by free-flow electrophoresis from C. roseus and other plant materials. The degree of sealing of the tonoplast and plasmamembrane vesicles was probed by their ability to pump protons (measurements of quinacrine quenching) and to generate a membrane potential (absorption spectroscopy of Oxonol VI). A critical evaluation of vesicles sidedness is presented.
RESUMEN
Calcium channel blockers of the phenylalkylamine family bind specifically to membranes and inhibit calcium uptake in carrot protoplast. LU 49888, an azido derivative of phenylalkylamine, behaves as its unmodified homolog in terms of affinity and specificity and therefore allows us to probe the receptor by photoaffinity labeling. Upon UV irradiation, a 75-kDa peptide was specifically labeled. Incubation of microsomes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a zwitterionic detergent, led to the solubilization of the LU 49888-binding protein. Electrophoretic analysis under denaturing conditions and gel filtration of the solubilized "receptor-ligand" complex show a 75-kDa peptide mainly located at the plasma membrane. Consequently the LU 49888-binding protein in plants differs significantly from its animal counterpart by its size and may be a primary target for external signal molecules.
RESUMEN
The conditions of malate transport were defined in tonoplast vesicles purified from a microsomal homogenate of Catharanthus roseus cells by preparative free-flow electrophoresis. Isolated vesicles exhibited malate transport when the membranes were prepared by grinding the cells in a homogenisation medium only buffered in the acidic pH range. By using vesicles energized artificially by an imposed pH gradient (acid interior), it was shown that malate is actively accumulated in response to the generation of a proton-motive force. Several lines of evidence (saturation kinetics, action of malate analogs and protein modifiers) support the concept that malate transport is mediated by a protein carrier which could be implicated in the uptake process as its protonated form. The malate transported in the vesicles was released by lowering the external malate concentration. The release was prevented by the anion transport inhibitor DIDS indicating the reversibility of the carrier.
Asunto(s)
Malatos/metabolismo , Orgánulos/metabolismo , Plantas/metabolismo , Transporte Biológico , Proteínas Portadoras/metabolismo , Sistema Libre de Células , Células Cultivadas , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Plantas/ultraestructuraRESUMEN
Tonoplast vesicles were purified from a microsomal fraction isolated from etiolated soybean hypocotyls (Glycine max L.) by preparative free-flow electrophoresis. Marker enzyme determinations and immunoblot analysis against the vacuolar-ATPase confirmed the nature and the purity of the isolated membranes. A purified tonoplast fraction also was obtained by consecutive sucrose and glycerol centrifugation which was further resolved into two different populations of vesicles (T(A) and T(B)) by free-flow electrophoresis. The determination of the sidedness of these different vesicles included concanavalin A binding as an imposed label, NADH-ferricyanide oxidoreductase cytochemistry, and ATPase latency. The tonoplast fractions, obtained by consecutive sucrose and glycerol gradient centrifugations, were found to consist of a mixture of two populations of vesicles of opposite sidedness. The least electronegative fraction obtained by free-flow electrophoresis (T(B)) consisted predominantly of cytoplasmic side out tonoplast vesicles while a fraction of greater electronegativity (T(A)) contained the cytoplasmic side in tonoplast vesicles. The relative amounts of each type of vesicle varied with the method of homogenization. Razor blade chopping, Polytron, and Waring Blendor homogenization gave predominantly cytoplasmic side out vesicles, whereas mashing with a mortar and pestle gave nearly equal amounts of the two populations of membrane vesicles of different orientation.
RESUMEN
Absolute orientations (sidedness) of plasma membrane vesicles obtained in highly purified fractions by preparative free-flow electrophoresis and by aqueous two-phase partition were determined based on ATPase latency and morphological criteria. Free-flow electrophoresis yielded two plasma membrane fractions. One, the least electronegative and designated fraction ;E,' was pure plasma membrane. The other, more electronegative and designated fraction ;C,' was heavily contaminated by various other cellular membranes. Plasma membrane vesicles from both fraction C and fraction E partitioned into the upper phase with aqueous two-phase partitioning. Purified plasma membrane obtained from microsomes by two-phase partition (upper phase) when subjected to free-flow electrophoresis also yielded two fractions, one fraction co-migrated with fraction C and another fraction co-migrated with fraction E. Both fractions exhibited an ATPase activity sensitive to vanadate and insensitive to nitrate and azide. ATPase activity was used as a structure-linked latency marker for the inner membrane surface. Concanavalin A binding (linked to peroxidase) was used as an imposed electron microscope marker for the outer membrane surface. Fraction E vesicles showed low ATPase latency (two-fold or less) and weak reactivity with concanavalin A peroxidase. In contrast, fraction C vesicles were characterized by much greater latencies upon detergent treatment (sevenfold) and a strong reaction with concanavalin A peroxidase. Two-phase partition as the initial procedure for plasma membrane isolation, yielded mixtures of vesicles of both inside out and right-side out orientation. Free-flow electrophoresis resolved the plasma membrane isolates into vesicles from fraction C which were right-side out (cytoplasmic side in), and vesicles from fraction E which were wrong-side out (cytoplasmic side out). Therefore, the two methods used in series, provided highly purified membrane preparations of apparently homogenous vesicles of opposite known absolute orientations.
RESUMEN
Proteases from mesophyll protoplasts of Melilotus alba were identified by standard proteolytic assays and separated using different chromatographic techniques. Their characterization also included their subcellular location. Besides the evidence for the multiplicity of the proteolytic enzymes, two protease sets were distinguished endopeptidases, which are exclusively vacuolar, and aminopeptidases, which are widely distributed throughout the cell. Cytosol-located enzymes were tested as substrates of the two sets of proteases, by studying comparatively the time-course changes of enzyme activities during incubation in total protoplast extracts, or in cytosol fractions devoid of vacuolar proteases. The degradation of phosphoenolpyruvate-carboxylase protein, a typical cytosolic enzyme, in the presence of purified amino-and endopeptidases, was also estimated by immunoprecipitation studies. Only the vacuolar endopeptidases are effective in the degradation of cytosolic enzymes. Hydrolytic enzyme activities mostly of vacuolar origin were very stable during incubation in total protoplast extracts. These proteins therefore appear to be particularly resistant to proteolytic attack. The results indicate that, in plants, the effective proteolytic system acting on cytosolic enzymes seems to be vacuole-located, and that the selectivity in protein degradation may be imposed by the susceptibility of the protein being degraded and by its transfer into the vacuoles.
RESUMEN
In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ([(14)C]p-fluorophenylalanine), were degraded more rapidly than normal ones ([(14)C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole.