Subcellular Localization of Sorbitol-6-phosphate Dehydrogenase in Protoplast from Apple Cotyledons

The subcellular localization of sorbitol-6-phosphate (S6P) dehydrogenasein protoplasts of apple cotyledons was examined by differentialcentrifugation and linear sucrose density gradient centrifugation(30–60%, w/w). The distribution of S6P dehydrogenase activitywas 55% in the 500 x g pellet of the homogenate and 35% in thesupernatant of 105,000 x g. When the x g pellet was recentrifugedin a linear sucrose density gradient, one major peak of activitywas found at a density of 1.23. This peak coincided with themajor peak of chlorophyll and NADP⁺-triose-P dehydrogenase activity.When the 500 x g pellet was sonicated, the major peak of S6Pdehydrogenase activity shifted to a lighter density (d=1.18).The shifted peak also coincided with the peak of chlorophyll.The enzyme detected in the major peak of chlorophyll (d=1.23)was partially solubilized by sonic or detergent treatment, butnot by hypotonic solution. The results supported the localizationof S6P dehydrogenase in chloroplasts, and presumably their associationwith thylakoid membranes. Part of the enzyme was assumed tobe naturally present in the cytosol, too. (Received November 4, 1980; Accepted January 21, 1981)     

ATP-Promoted Sorbitol Transport into Vacuoles Isolated from Apple Fruit

Sorbitol was transported actively into vacuoles isolated fromapple (Malus pumilla Mill, var domestica Schneid.) fruit flesh.The uptake was stimulated up to twofold by the addition of ATP,and the ATP dependent uptake showed a saturation curve as tothe substrate concentration. The optimum uptake of sorbitolwas pursued in the acidic range of pH 5 to 6. The K_m value forthe ATP dependent sorbitol uptake was about 5 mM. Sorbitol uptake was clearly inhibited by PCMB and uncouplers(CCCP and DCCD), and to a lesser extent by orthovanadate, butonly slightly by oligomycin. K⁺ stimulated sorbitol uptake.Sorbitol was converted to other sugars (glucose) only very slowlywhen transported across the tonoplast. This suggests that sorbitolis transported into vacuoles by a carrier mediated transportsystem coupled with H⁺- ATPase, localized on the tonoplast.Sucrose uptake into the vacuoles was also enhanced by ATP. (Received May 31, 1986; Accepted March 2, 1987)     

Distribution of Sorbitol, Neutral Sugars, Free Amino Acids, Malic Acid and Some Hydrolytic Enzymes in Vacuoles of Apple Cotyledons

Vacuoles of apple cotyledons (Malus pumila Mill. var. domesticaSchneid.) were obtained by purification with Ficoll densitygradient centrifugation after the protoplasts were lysed byboth osmotic shock and the addition of EDTA. High levels ofacid protease and carboxypeptidase activity were detected inthe vacuoles along with acid phosphatase, phosphodiesteraseand ATPase. The distribution of sorbitol and other sugars inthe vacuoles, the protoplast and extracellular free space wasdetermined. About 45, 60 and 90% of the sorbitol, glucose andsucrose, respectively, contained in whole tissue were foundin the extracellular free space, and 54% of the sorbitol inthe protoplast was detected in the vacuole. The sorbitol inthe vacuoles and the extravacuole of the protoplast was about80 and 70%, respectively, of the total sugar content of thecell. Arginine was the most abundant free amino acid in theprotoplast, and about 90% of it was contained in the vacuole.More than 80% of the total amino acids and 50% of the proteinwere also located in the vacuole, as well as most of the malate.The amounts of the total sugars, total amino acids, proteinand malate in the vacuoles were to 250, 400, 48 and 9 µg/10⁶cells, respectively. The results suggest that the vacuoles ofapple cotyledons contain a large pool of amino acids and proteinsrather than sugars, and have a close connection with proteinbody degradation. ¹ This paper is contribution A-l37 of the Fruit Tree ResearchStation. (Received January 20, 1982; Accepted May 18, 1982)     

Subcellular Localization of IAA Oxidase in Peas

Indoleacetic acid (IAA) oxidase has been reported to be involved in plant growth because of its alleged role in the control of endogenous IAA levels. This purported role was reevaluated in terms of the properties and subcellular location of the enzyme in etiolated pea (Pisum sativum L. var. Alaska) epicotyls.     

Accumulation and Subcellular Localization of alpha-Galactosidase-Hemagglutinin in Developing Soybean Cotyledons

We have investigated the accumulation and intracellular localization of soybean (Glycine max [L.] Merr. cv Forrest) α-galactosidase-hemagglutinin during seed development. Cotyledon tissue was embedded in Lowicryl K4M and immunocytochemical localization was accomplished through treating thin sections with α-galactosidase antisera followed by indirect labeling with protein A coupled to colloidal gold. Gold particles were localized on the Golgi apparatus and protein bodies. We interpret this to indicate that α-galactosidase-hemagglutinin is transferred to and transported through the Golgi apparatus and finally deposited within the protein body by a Golgi apparatus-mediated process.     

Subcellular Localization and Developmental Changes of Aspartate-alpha-Ketoglutarate Transaminase Isozymes in the Cotyledons of Cucumber Seedlings

The total activity of aspartate-α-ketoglutarate transaminase in the cotyledons of cucumber (Cucumis sativus L.) seeds increased 17-fold during the first 2 days of germination in darkness and then declined gradually to 20% of the peak activity after 10 days. Exposure of the seedlings to light at day 3 accelerated the decline. The enzyme in the cotyledon extracts from seedlings at various ages was resolved into six distinct isozymes by starch gel electrophoresis. Isozymes 1 and 2 were glyoxysomal isozymes with different developmental patterns. Isozyme 1 followed the developmental pattern of the total enzyme activity in darkness, and was rapidly eliminated upon illumination. Isozyme 2 increased in activity to a peak at day 2 and declined rapidly thereafter, and disappeared completely at day 6; this developmental pattern was independent of light. No major difference in the optimal pH for activity, substrate specificity, and reversibility was observed between isozymes 1 and 2. The combined developmental pattern of isozymes 1 and 2 during germination correlated with that of the glyoxysomes. Isozyme 3 was located in the cytosol and its developmental pattern followed that of the total activity. Isozymes 4,5, and 6 were plastid isozymes and appeared only after 2 days of germination. Unlike many other chloroplast enzymes, the appearance of the chloroplast transaminase isozymes was under temporal control and was independent of illumination. No enzyme activity was detected in isolated mitochondria. The findings illustrate a complicated cellular control system for the appearance of various organelle-specific transaminase isozymes and thus the amino acid metabolism during germination.     

Subcellular Localization of Glycosyl Transferases Involved in Glycoprotein Biosynthesis in the Cotyledons of Pisum sativum L

Subcellular membrane fractions from 21-day-old pea (Pisum sativum) cotyledons that have associated UDP-N-acetylglucosamine N-acetylglucosaminyl transferase and GDP-mannose mannosyl transferase activities have been isolated and identified. The rough endoplasmic reticulum (RER) is the principal location of glycosyl transferases involved in the assembly of lipid-linked sugar intermediates and glycoproteins. Antimycin A-insensitive NADH-cytochrome c reductase activity was used to identify RER at a density of 1.165 g/cc in sucrose gradients. The high proportion of RER in this fraction was confirmed by electron microscopy.

Other mannosyl transferases are found at a density of 1.123 g/cc and 1.201 g/cc but these glycosyl transferases do not appear to be involved with the formation of lipid-linked sugar intermediates utilized in glycoprotein biosynthesis.

     

Stimulation of the Uptake of Sorbitol into Vacuoles from Apple Fruit Flesh by Abscisic Add and into Protoplasts by Indoleacetic Acid

The uptake of sorbitol into vacuoles from immature flesh ofapple fruit (Maluspumila Mill, var domestica Schneid.) was facilitatedby 10^–6 M ABA, while such uptake into protoplasts wasnot stimulated. By contrast, the application of 10^–5 MIAA facilitated uptake of sorbitol into protoplasts but didnot significantly into vacuoles. (Received July 17, 1990; Accepted December 25, 1990)     

Energy Coupled Transport of Sorbitol and Other Sugars into the Protoplast Isolated from Apple Fruit Flesh

The uptake kinetics of sorbitol, sucrose, glucose and fructoseacross the plasma membrane using protoplasts isolated from applefruit flesh (Malus pumila Mill. var. domestica Schneid.) wasinvestigated. When sorbitol was taken up into the cell, PCMBS-sensitivesaturable transport was distinguishable from the diffusive transport.At a low sorbitol concentration, the saturable transport systemaccounted for more than 50% of the total uptake, whereas ata high concentration the diffusive transport system was moredominant. The saturable transport was suggested be a carrier-mediatedtransport system coupled with ATP because the system was inhibitedCCCP or orthovanadate. The K_m value for sorbitol was computedto be 3.6mM. A carrier-mediated transport system coupled withATP was also observed for glucose and fructose with correspondingK_m values of 5.0 and 2.5 mM. However, no saturable transportfor sucrose was observed over a range of 0.1 to 10 mM sucroseconcentration. The relationship among these sugar transportsystems across the plasma membrane, apoplastic unloading, andsugar accumulation vacuoles are discussed. ¹Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received April 8, 1988; Accepted June 8, 1988)     

Organic Acid Metabolism in Cellular Organelles of Vigna cylindrica (Mitorisasage) Cotyledons

In starchy cotyledons of Vigna cylindrica (L.) Skeels (Mitorisasage)during seed germination, the enzymes of the glyoxylate cyclewere located in the matrix of mitochondria. Glyoxysomes wereabsent. The glyoxylate cycle in the mitochondria supplies organicacids to the tricarboxylic acid cycle. In mitochondria, isocitratelyase activity was much higher than malate synthase activity.Part of the glyoxylate thus produced in mitochondria may benonenzymatically converted to formate by H₂O₂ and the formatethen converted to CO₂ by peroxidase or by formic dehydrogenase.The activity of superoxide dismutase, which supplies H₂O₂, washigher in mitochondria than in peroxisomes. The remaining glyoxylatein mitochondria is possibly converted to glycine by alanine-glyoxylateaminotransferase or transported to peroxisomes which lackedisocitrate lyase activity but had high malate synthase activity.In peroxisomes, glyoxylate may be also produced from urate,as is suggested by the fairly high activities of uricase, allantoinaseand allantoicase. Judging from the enzyme distribution, Vignaperoxisomes should be capable of producing malate, oxalacetate,citrate, isocitrate and a-ketoglutarate. ¹Present address: Department of Horticulture, College of Agricultureand Animal Science, Yeugnam University, Gyeongsan 632, Korea. (Received May 27, 1987; Accepted October 7, 1987)     

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

The endoplasmic reticulum (ER) is a large, continuous membrane-bound organelle comprised of functionally and structurally distinct domains including the nuclear envelope, peripheral tubular ER, peripheral cisternae, and numerous membrane contact sites at the plasma membrane, mitochondria, Golgi, endosomes, and peroxisomes. These domains are required for multiple cellular processes, including synthesis of proteins and lipids, calcium level regulation, and exchange of macromolecules with various organelles at ER-membrane contact sites. The ER maintains its unique overall structure regardless of dynamics or transfer at ER-organelle contacts. In this review, we describe the numerous factors that contribute to the structure of the ER.The endoplasmic reticulum (ER) is a dynamic organelle responsible for many cellular functions, including the synthesis of proteins and lipids, and regulation of intracellular calcium levels. This review focuses on the distinct and complex morphology of the ER. The structure of the ER is complex because of the numerous distinct domains that exist within one continuous membrane bilayer. These domains are shaped by interactions with the cytoskeleton, by proteins that stabilize membrane shape, and by a homotypic fusion machinery that allows the ER membrane to maintain its continuity and identity. The ER also contains domains that contact the plasma membrane (PM) and other organelles including the Golgi, endosomes, mitochondria, lipid droplets, and peroxisomes. ER contact sites with other organelles and the PM are both abundant and dispersed throughout the cytoplasm, suggesting that they too could influence the overall architecture of the ER. As we will discuss here, ER shape and distribution are regulated by many intrinsic and extrinsic forces.     

Automated Identification of Subcellular Organelles by Coherent Anti-Stokes Raman Scattering

Coherent anti-Stokes Raman scattering (CARS) is an emerging tool for label-free characterization of living cells. Here, unsupervised multivariate analysis of CARS datasets was used to visualize the subcellular compartments. In addition, a supervised learning algorithm based on the “random forest” ensemble learning method as a classifier, was trained with CARS spectra using immunofluorescence images as a reference. The supervised classifier was then used, to our knowledge for the first time, to automatically identify lipid droplets, nucleus, nucleoli, and endoplasmic reticulum in datasets that are not used for training. These four subcellular components were simultaneously and label-free monitored instead of using several fluorescent labels. These results open new avenues for label-free time-resolved investigation of subcellular components in different cells, especially cancer cells.     

Automated Identification of Subcellular Organelles by Coherent Anti-Stokes Raman Scattering

Coherent anti-Stokes Raman scattering (CARS) is an emerging tool for label-free characterization of living cells. Here, unsupervised multivariate analysis of CARS datasets was used to visualize the subcellular compartments. In addition, a supervised learning algorithm based on the “random forest” ensemble learning method as a classifier, was trained with CARS spectra using immunofluorescence images as a reference. The supervised classifier was then used, to our knowledge for the first time, to automatically identify lipid droplets, nucleus, nucleoli, and endoplasmic reticulum in datasets that are not used for training. These four subcellular components were simultaneously and label-free monitored instead of using several fluorescent labels. These results open new avenues for label-free time-resolved investigation of subcellular components in different cells, especially cancer cells.     

NADP+-Dependent Sorbitol Dehydrogenase Found in Apple Leaves

An NADP⁺-dependent sorbitol dehydrogenase that catalyzes sorbitoland glucose was found in apple leaves. The partially purifiedenzyme had optimum activity at pH 9.6 and a K_m value of 128mM for sorbitol. Among the polyols studied, this enzyme showedthe most activity for sorbitol. ¹This paper is contribution A-173 of the Fruit Tree ResearchStation. (Received June 4, 1984; Accepted July 31, 1984)     

Protein Subcellular Localization in Bacteria

Like their eukaryotic counterparts, bacterial cells have a highly organized internal architecture. Here, we address the question of how proteins localize to particular sites in the cell and how they do so in a dynamic manner. We consider the underlying mechanisms that govern the positioning of proteins and protein complexes in the examples of the divisome, polar assemblies, cytoplasmic clusters, cytoskeletal elements, and organelles. We argue that geometric cues, self-assembly, and restricted sites of assembly are all exploited by the cell to specifically localize particular proteins that we refer to as anchor proteins. These anchor proteins in turn govern the localization of a whole host of additional proteins. Looking ahead, we speculate on the existence of additional mechanisms that contribute to the organization of bacterial cells, such as the nucleoid, membrane microdomains enriched in specific lipids, and RNAs with positional information.Our view of the organization of the bacterial cell has changed radically over the past two decades. Once seen as an amorphous vessel harboring a homogeneous solution of proteins, these primitive organisms are now known to have an intricate subcellular architecture in which individual proteins localize to particular sites in the cell, often in a dynamic manner. Of course, bacteria frequently show conspicuous morphological features, such as division septa, flagella, pili, and stalks, which implied a nonuniform, underlying distribution of proteins. But it was not until the early 1990s that it became clear that proteins can, and often do, have distinctive subcellular addresses. Among the earliest discoveries were: (1) the formation of a ringlike structure at the mid cell position by the cytokinetic protein FtsZ (Bi and Lutkenhaus 1991), (2) the clustering of chemotaxis proteins at the poles of cells (Alley et al. 1992), (3) the compartment-specific production of sporulation proteins and their assembly into shell-like structures (Driks and Losick 1991), and (4) the asymmetric distribution of proteins involved in actin polymerization along the cell surface (Goldberg et al. 1993; Kocks et al. 1993). These discoveries were initially made by immunoelectron and immunofluorescence microscopy with fixed cells, but the discovery of green fluorescent protein (GFP) and the demonstration that proteins could retain their proper subcellular localization as GFP fusions opened the way to visualizing proteins and their dynamic behavior in living cells, including, importantly, in bacteria (Arigoni et al. 1995).Knowing where proteins are in the cell is often critical to understanding their function. Thus, the position of the aforementioned FtsZ ring (the Z-ring) dictates where cytokinesis will take place (Margolin 2005). The clustering of chemotaxis proteins plays an important role in the extraordinary gain in the responsiveness of chemotatic behavior to small changes in attractants (Ames and Parkinson 2006). Where sporulation proteins are produced and the way in which they assemble governs spore morphogenesis (Stragier and Losick 1996; Errington 2003). The asymmetric distribution of actin-polymerization proteins on the cell surface explains how certain pathogens harness host cytoskeletal proteins for their own motility (Smith et al. 1995). From these and other examples emerge a view of the bacterial cell as a dynamic, three-dimensional system in which protein localization and changes in protein localization over time orchestrate growth, the cell cycle, behavior, and differentiation.Here, after an initial discussion of general principles governing the positioning of proteins within the cell, we consider five broad categories of subcellular localization: the divisome, polar assemblies, cytoplasmic clusters, cytoskeletal elements, and organelles. We end by looking ahead to exciting new aspects of bacterial cytology just emerging from current research. Our goal is not to be comprehensive but rather to focus on examples that are illustrative of general principles of protein localization. Comprehensive treatment of individual topics can be found in other articles on this topic.     

Sorbitol in Tracheal Sap of Apple as Related to Temperature

The influence of dormant-season temperatures on levels of sorbitol was determined in an effort to provide further information on the possible role of sorbitol in dormancy of apple tress (Malus sylvestris Mill.). Two-year-old shoots were collected throughout the dormant season; sorbitol and sugars were determined in tracheal sap extracted under vacuum and in ground dried wood extracted in a soxhlet apparatus. Sorbitol and sugar trimethylsilyl derivatives were detected by gas chromatography. Levels of sorbitol in the sap generally increased during subfreezing temperatures and decreased during warm periods throughout the dormant season. Early peak increases in sap sorbitol appeared to coincide with the beginning of leaf senescence. Postharvest levels of reducing sugars, sorbitol, and particularly sucrose in the wood, increased as the temperature decreased during the dormant season, and the sugar levels decreased with warming temperatures in the spring. Our data indicate that sorbitol and sucrose are important reserves of storage carbohydrates in resting apple trees.     

Localization and Substrate Specificity of Glycosidases in Vacuoles of Nicotiana rustica

Vacuoles isolated from Nicotiana rustica var brasilia have been shown to contain significant levels of glycosidase activity when assayed using p-nitrophenyl-glycosides as substrates. The substrate specificity for the glycosidases in the vacuolar fraction closely paralleled that found in the protoplasts, and the leaf tissue from which the vacuoles were isolated. The substrate specificity of the vacuolar enzyme(s) was different from glycosidic activity found in the commercial digestive enzyme preparations used to isolate the protoplasts from leaf tissue. It was demonstrated that 70 to 90% of the glycosidases that were found in the protoplasts appeared to be localized within the vacuole, when the p-nitrophenyl substrates α- and β-;d-galactose, β-d-glucose, and α-d-mannose were used. Neither the vacuolar nor the protoplast enzymes were active towards the naturally occurring phenolic glycoside, rutin. α-Mannosidase appears to be a valuable marker enzyme for vacuoles isolated from mesophyll leaf cells of tobacco.     

Purification and Properties of NAD-Dependent Sorbitol Dehydrogenase from Apple Fruit

This is the first report of the purification of NAD-dependentsorbitol dehydrogenase (NAD-SDH) from a plant source. The enzymewas extracted from apple (Malus domestica cv. Ourin) fruit andpurified until it appeared as a single polypeptide chain ona gel after SDS-PAGE. From the apparent molecular mass of 62kDa obtained by SDS-PAGE and that of 120 kDa by gel filtration,the enzyme appeared to be a homodimer. Maximum rates of oxidationof sorbitol and reduction of fructose were observed at pH 9.6and pH 6.0, respectively. The K_m for oxidation of sorbitol was40.3 mM and that for reduction of fructose was 215 mM. The maximumrate of oxidation of sorbitol was about 10 times higher thanthat of the reduction of fructose. The results of the kineticanalysis strongly suggest that in vivo the enzyme would favorthe conversion of sorbitol to fructose over the reverse reaction.None of the divalent cations tested had any effect on the oxidationof sorbitol by NAD-SDH. The reaction catalyzed by NAD-SDH wasnot specific to sorbitol and other substrates could also beoxidized. Among the tested substrates, ethyl alcohol had a particularlyhigh affinity for the enzyme. (Received February 2, 1994; Accepted May 31, 1994)     

Ultrastructure of Blepharoplast and Other Organelles of the Spermatid in Ginkgo biloba

Both blepbaroplast and osmiophilic globule were characteristic structures to the spermatid of Ginkgo biloba. The blepharoplast of Ginkgo biloba ranged from 3 ～ 4 μm in diameter and consisted of a number of basal centrioles radiating from an electron dense core that contained electron-lucent areas with microtubule structure. Microtubules extended radially from the blepharoplast into the cytoplasm. A large round osmiopbilie globule with a diameter of about 10～20/μm, was located between the blepharoplast and the nucleus, while a filbrillogranular body in the cytoplasm was opposite to the osmiophilic globule. There were numerous mitochondria, plastids, endoplasmic reticulia and dictiosomes in the cytoplasm, particularly around the blepharoplast and the osmiophilic globule of sperm cells. The nucleus of spermatid in Ginkgo biloba was large and roundly elliptical in shape. The large spheroidal nucleolus was the most obvious structure in the nucleus, There were two regions in the nucleolus distinguished by TEM: A ring-shaped granular component, which contained maturing ribosomal precursor particles; and a centrally placed fibrillar component. The nuclear pore complexes in the nuclear envelope were plentiful but not evenly distributed.