Biochemical, immunological, and immunocytochemical evidence for the association of chalcone synthase with endoplasmic reticulum membranes.

Abstract

Chalcone synthase [naringenin-chalcone synthase; malonyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing), E.C. 2.3.1.74], the key enzyme of flavonoid pathways that was believed to be soluble, has been localized on ribosome-bearing endoplasmic reticulum membranes in the epidermis of buckwheat (Fagopyrum esculentum M.) hypocotyls. Enzyme activity measurement and immunoblots of buckwheat hypocotyl homogenates that were fractionated on linear sucrose density gradients and developed with a specific chalcone synthase antibody and a 20-nm ImmunoGold coijugate showed the presence of chalcone synthase in fractions enriched in endoplasmic reticulum membranes. The presence of chalcone synthase on these membranes was not caused by nonspecific adsorption or entrapment of proteins. Immunocytochemical investigations with both a 5-nm and a 20-nm ImmunoGold conjugate showed that chalcone synthase was associated with the cytoplasmic face of rough (ribosome bearing) endoplasmic reticulum membranes. Plasma membrane, nucleus, plastids, mitochondria, golgi, and the tonoplast were not labeled. These data are consistent with our earlier described model suggesting that the synthesis of phenylpropanoids and flavonoids takes place partially or fully on membrane-associated enzyme complexes.Flavonoid compounds are produced by most, if not all, higher plants and are synthesized from p-coumaryl-CoA and malonyl-CoA by the enzyme chalcone synthase [CHS; naringenin-chalcone synthase; malonyl-CoA:4-coumaroyl-CoA malonyltransferase (cyclizing), EC 2.3.1.74]. Cell fractionation experiments showed that the majority of the activity of this enzyme was "soluble," with small but significant portions associated with a membrane fraction (1). Ultracentrifugation (2) and gel-filtration experiments (3) suggested that CHS is associated with the endoplasmic reticulum (ER) membranes. Convincing evidence exists that another soluble enzyme of this pathway, phenylalanine ammonia lyase (E.C. 4.3.1.5) is at least in part membrane associated (4).Detection of enzyme activities in the soluble cytoplasmic fraction after cell fractionation is not a convincing argument for subcellular localization in vivo. Most homogenization procedures destroy cellular organization, and most suspension media that are used during cell fractionations are not physiological in nature. It is becoming increasingly clear that many so-called cytosolic enzymes interact with each other and the structural components of the cell, and the enzyme activities are modified by these interactions (5). Thus, the term "soluble enzyme" should not be taken as indicating that the catalytically active compartment is a true solution. Rather, it should be regarded as an operational definition, referring more to the result of homogenization and centrifugation procedures in subcellular fractionation than to any established in vivo characteristic.Our previous studies on localizing weakly associated enzymes or ER membranes by activity alone were greatly hampered by inactivation of enzymes during the isolati...

Sporopollenin Biosynthetic Enzymes Interact and Constitute a Metabolon Localized to the Endoplasmic Reticulum of Tapetum Cells

“…The occurrence of multienzyme complexes in phenylpropanoid and flavonoid pathways is believed to explain how common intermediates in these pathways may lead to distinct metabolites. Different experimental approaches such as radioactive labeling, cell fractionation, or microscopic studies have suggested that enzyme complexes are structured on the ER, resulting in the channeling into a specific enzymatic reaction chain leading to a given final product (Czichi and Kindl, 1977;Hrazdina et al, 1987;Burbulis and Winkel-Shirley, 1999;Chen et al, 2011a). Distinct cytochrome P450 hydroxylases are involved in those pathways and serve as anchors of the metabolons to the ER membrane, as is likely the case for the sporopollenin metabolon evidenced in the present work.…”

Genetic regulation and photocontrol of anthocyanin accumulation in maize seedlings.

“…As shown in Figure 8, this antibody recognizes a single protein species of approximately 42 kD in both -g and -r lines. This matches the size of chalcone synthase proteins isolated from a number of sources (Mol et al, 1983;Rail and Hemleben, 1984;Hrazdina, Zobel, and Hoch, 1987;Schmelzer, Jahnen, and Hahlbrock, 1988). The maximum signal is obtained from the soluble protein fraction of -r and -g plants (lanes 3 and 4), although low levels can be detected in the microsomal fractions (lanes 1 and 2).…”

Reconstitution of the Entry Point of Plant Phenylpropanoid Metabolism in Yeast (Saccharomyces cerevisiae)

“…It has been shown that a specific PAL isoform (PAL1) is physically associated with both isolated microsomes and the cytoplasmic fraction in fractionated tobacco cell extracts, supporting a model in which PAL associates with MECs in an isoform-specific manner to modulate a carbon flux into specific downstream pathways (12). Other data from experiments employing classical approaches such as double radioactive labeling assays (13)(14)(15), enzyme co-purification (15,16), and immunohistochemical localization (17) support the presence of MECs among enzymes in the entry point or downstream pathways. Investigation of possible MECs in phenylpropanoid metabolism has been further pursued in recent years using various molecular techniques, revealing direct proteinprotein interactions among enzymes in the flavonoid pathway in Arabidopsis (18) and the elicitor-dependent endoplasmic reticulum (ER)-localization of an otherwise cytosolic isoflavone Omethyltransferase in alfalfa (19).…”