In 1898, Camillio Golgi reported a new cellular constituent with the form of an extensive intracellular network (the apparato reticolare interno), which now bears his name. However, the history of Golgi's apparatus is replete with controversy regarding its reality, what components of the cell should be included under its aegis, and what terminology should be used when referring to it. Electron microscopy has resolved many of these controversies and it is appropriate that this volume emphasize that aspect of Golgi apparatus discovery. The principal structural component of the Golgi apparatus is the stack of cisternae, or dictyosome. As determined both biochemically and at the level of electron microscopy, the dictyosome is a highly ordered and polarized structure. The maintenance of order within the stack is thought to result from either intercisternal bonding constituents, or filamentous structures (or both) that bridge the space between adjacent cisternae. Mechanisms proposed for movement of membrane and product into and out of the dictyosome (i.e., the Golgi apparatus stack) include a serial mode which functions exclusively by the formation, displacement, and loss of cisternae from the stack, and a parallel mode which functions exclusively by the movement of membrane, product, or precursor molecules directly into the peripheral edges of the cisternae. In the parallel mode, all cisternae can be accessed either singly or simultaneously, at least in theory, at any position within the stack. It is probable that both the serial and the parallel modes function concomitantly and need not be mutually exclusive. Finally, the peripheral tubules of the cisternae represent a major membranous constituent of the cell with potentially unique functions. These tubules interconnect cisternae of adjacent stacks and may represent the major site of receptors for the shuttle (i.e., parallel) type of transfer among cisternae. Peripheral tubules as extensions of the cisternal lumina into the cytoplasm presumably have other functions, but these, like the tubules themselves, have only rarely been accommodated into functional models of Golgi apparatus dynamics in secretion or membrane flow.
A procedure is described whereby highly purified fractions of plasma membrane and tonoplast were isolated from hypocotyls of dark-grown soybean (Glycine max L. var Wayne) by the technique of preparative free-flow electrophoresis. Fractions migrating the slowest toward the anode were enriched in thick (10 nanometers) membranes identified as plasma membranes based on ability to bind N-1-naphthylphthalamic acid (NPA), glucan synthetase-II, and KV-stimulated, vanadate-inhibited Mg2e ATPase, reaction with phosphotungstic acid at low pH on electron microscope sections, and morphological evaluations. Fractions migrating farthest toward the anode (farthest from the point of sample injection) were enriched in membrane vesicles with thick (7-9 nanometers) membranes that did not stain with phosphotungstic acid at low pH, contained a nitrate-inhibited, Cl-stimulated ATPase and had the in situ morphological characteristics of tonoplast including the presence of flocculent contents. These vesicles neither bound NPA nor contained levels of glucan synthetase II above background. Other membranous cell components such as dictyosomes (fucosyltransferase, latent nucleosidediphosphate phosphatase), endoplasmic reticulum vesicles (NADH-and NADPHcytochrome c reductase), mitochondria (succinate-2(p-indophenyl)-3-pnitrophenyl)-5-phenyl tetrazolium-reductase and cytochrome oxidase) and plastids (carotenoids and monogalactosyl diglyceride synthetase) were identified on the basis of appropriate marker constituents and, except for plastid thylakoids, had thin (<7 nanometers) membranes. They were located in the fractions intermediate between plasma membrane and tonoplast after free-flow electrophoretic separation and did not contaminate either the plasma membrane or the tonoplast fraction as determined from marker activities. From electron microscope morphometry (using both membrane measurements and staining with phosphotungstic acid at low pH) and analysis of marker enzymes, both plasma membrane and tonoplast fractions were estimated to be about 90% pure. Neither fraction appeared to be contaminated by the other by more than 3%.With elongating plant cells, the total membrane surface contributed by tonoplast surrounding the central vacuole and the I
A phosphatidylinositol-specific phospholipase C of plant stems (EC 3.1A.10) assayed at pH 6.6 and at 30°C cleaved phosphatidylinositol such that more than 85% of the product was inositol-l-phosphate. Other phospholipids were cleaved 5 to 10% or less under these conditions. The phospholipase had both a soluble and a membrane-associated form. The soluble activity accounted for approximately 85 to 90% of the activity and 15% was associated with membranes. The membrane-associated activity was most concentrated in the plasma membranes of hypocotyl segments of both soybean (Glycine max) and bushbean (Phaseolus vulgaris). The plasma membrane location was verified by analysis of highly purified plasma membranes prepared both by aqueous two-phase partitioning and by preparative free-flow electrophoresis and from the quantitation of the activity in all maior cell fractions. Internal membranes also contained phospholipase C activity but at specific activity levels of about 0.1 those present in plasma membranes. Golgi apparatus-enriched fractions from which plasma membrane contaminants were removed by twophase partition contained the activity at specific activity levels 0.2 those of plasma membrane. Both the soluble and the membrane-associated activity was stimulated by calcium but not by calmodulin, either alone or in the presence of calcium.There have been periodic reports of soluble phospholipase C activities in plants (8,9,11). Additionally, breakdown of membrane-associated phosphatidylinositol has been demonstrated based on analyses using radiolabeled phosphatidylinositol (5,8,9,20). The recent interest in phospholipases has been stimulated by their importance in signal-response transduction mechanisms (3,18,22) and the occurrence of phosphoinositol phosphates in plants (4,7,21,25). The latter in carrot cells appear concentrated in plasma membranes (JJ Wheeler, WF Boss, personal communication). In this paper we have examined the subcellular distribution of a phosphatidylinositol-specific phospholipase C of etiolated hypocotyls of soybean and bushbean and the response of the membrane-associated and soluble forms of the activity to calcium and calmodulin. The findings show a clear plasma membrane location of the membrane-associated form of the enzyme and a stimulation ofboth the soluble and the membraneassociated forms of the enzyme with calcium but not with calmodulin. The calcium-stimulated activity appears to be present in very low levels throughout the system of internal membranes as well. ' Supported in part by a grant 8206222 from the National Science Foundation (to D.J.M.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.