Here we show that scavenger receptor class B type I is present in the small-intestine brush border membrane where it facilitates the uptake of dietary cholesterol from either bile salt micelles or phospholipid vesicles. This receptor can also function as a port for several additional classes of lipids, including cholesteryl esters, triacylglycerols, and phospholipids. It is the first receptor demonstrated to be involved in the absorption of dietary lipids in the intestine. In liver and steroidogenic tissues, the physiological ligand of this receptor is high-density lipoprotein. We show that binding of high-density lipoprotein and apolipoprotein A-I to the brush border membrane-resident receptor inhibits uptake of cholesterol (sterol) into the brush border membrane from lipid donor particles. This finding lends further support to the conclusion that scavenger receptor BI catalyzes intestinal cholesterol uptake. Our findings suggest new therapeutic approaches for limiting the absorption of dietary cholesterol and reducing hypercholesterolemia and the risk of atherosclerosis.

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A novel plant phospholipase D (PLD; EC 3.1.4.4) activity, which is dependent on phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and nanomolar concentrations of calcium, has been identified in Arabidopsis. This report describes the cloning, expression, and characterization of an Arabidopsis cDNA that encodes this PLD. We have designated names of PLD␤ for this PIP 2 -dependent PLD and PLD␣ for the previously characterized PIP 2 -independent PLD that requires millimolar Ca 2؉ for optimal activity. The PLD␤ cDNA contains an open reading frame of 2904 nucleotides coding for a 968-amino acid protein of 108,575 daltons. Expression of this PLD␤ cDNA clone in Escherichia coli results in the accumulation of a functional PLD having PLD␤, but not PLD␣, activity. The activity of the expressed PLD␤ is dependent on PIP 2 and submicromolar amounts of Ca 2؉ , inhibited by neomycin, and stimulated by a soluble factor from plant extracts. Sequence analysis reveals that PLD␤ is evolutionarily divergent from PLD␣ and that its N terminus contains a regulatory Ca 2؉ -dependent phospholipid-binding (C2) domain that is found in a number of signal transducing and membrane trafficking proteins.Phospholipase D (PLD; EC 3.1.4.4) 1 -catalyzed hydrolysis of glycerophospholipids produces phosphatidic acid (PA) and a hydrophilic constituent. This activity was first identified in plants and since has been found in animals and microorganisms. PLD in plants was originally proposed to be important in phospholipid catabolism, initiating a lipolytic cascade in membrane deterioration during senescence and stress injuries (1, 2). Recent studies in plants, animals, and yeast indicate that PLD hydrolysis plays a pivotal role in transmembrane signaling and cellular regulation (3-9). Activation of PLD has been proposed to mediate many cellular processes including cell proliferation, membrane trafficking, meiosis, and responses to external and internal stimuli. It has been suggested that multiple forms of PLD are involved in these diverse cellular processes since several studies have shown the presence of PLD variants that are expressed differently (9 -12). In castor bean (9) and rice (12), one PLD variant is constitutive whereas the appearance of other variants is associated with specific conditions such as rapid growth, wounding, and senescence. A distinct property shared by these variants is their in vitro requirement of millimolar Ca 2ϩ concentrations for optimal activity. Further analyses of the castor bean PLD variants have led to the suggestion that the catalytic activity of these variants results from the same gene product (9 -11).A recent study has provided important evidence for the presence of two plant PLDs that are derived from different gene products and regulated distinctly (13). One PLD requires polyphosphoinositides and submicromolar concentrations of Ca 2ϩ for activity and the other is PIP 2 -independent and is most active in the presence of millimolar amounts of Ca 2ϩ . The latter is the prevalent form of PLD that has been purified and cha...

Multiple molecular forms of phospholipase D (PLD; EC 3.1.4.4) were identified and partially characterized in endosperm of germinated seeds and leaves of castor bean (Ricinus communis L. var Hale). The different PLD forms were resolved by nondenaturing polyacrylamide gel eledrophoresis, isoelectric focusing, and sizeexclusion chromatography. PLD was deteded with both a PLD adivity assay and immunoblots with PLD-specific antibodies. There were three major forms of PLD, designated types 1,2, and 3, based on their mobility during nondenaturing polyacrylamide gel electrophoresis. Molecular masses of the PLD variants were estimated at 330, 230, and 270 k D for the types 1, 2, and 3, respedively. lsoeledric points of the native type 1, 2, and 3 PLDs were approximately 6.2,4.9, and 4.8. Under the in vitro assay conditions used, the three forms of PLD exhibited the same substrate specificity, hydrolyzing phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PC) but not phosphatidylserine (PS) and phosphatidylinositol (Pl). The three forms of PLD differed in their substrate preferences, and the order of activities was: PLD 1, PE > PC = PC; PLD 2, PE > PC > PC; PLD 3, PE = PC = PC. The K,,, values of PLDs 1,2, and 3 for PC were 1.92,2.62, and 5.18 mM, respectively. These PLDs were expressed differentially following seed germination and during leaf development. Type 1 was found in the early stages of seedling growth and in young leaves, type 2 was present in all the tissues and growth stages examined, and type 3 was expressed in senescent tissues. The PLDs shifted from largely cytosolic to predominantly membrane-associated forms during leaf development. The present studies demonstrate the structural heterogeneity of plant PLD and growth stage-specific expression of different molecular forms. The possible role for the occurrence of multiple molecular forms of PLD in cellular metabolism i s discussed.

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A cDNA from a novel Ca 2؉ -dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca 2؉ , undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca 2؉ which was bound with high affinity (K d Ϸ 0.37 M), the apparent Hill coefficient for Ca 2؉ -induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP͞ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca 2؉ binding N-terminal half of the transporter faces the cytosol.Peroxisomes and mitochondria are the two organelles present in mammalian eukaryotic cells for which an evolutionary endosymbiotic origin has been proposed (1, 2). Both organelles share a set of similar metabolic pathways, are the site of cellular oxygen consumption, and have similar macromolecular components and membrane phospholipid compositions (3). Mitochondria, in contrast to peroxisomes contain their own DNA and are surrounded by a double membrane rather than a single one. Despite the existence of mitochondrial DNA, most proteins are coded for by nuclear genes, synthesized on free cytosolic polysomes and subsequently posttranslationally sorted to their final mitochondrial location (for a review, see ref. 4). In principle, the same applies for peroxisomal proteins although much less is known about protein import in this organelle (for a review, see ref. 5).The mitochondrial inner membrane contains three different major classes of proteins: the electron transport chain complex, the ATP synthase complex, and the mitochondrial solute carriers. The first two complexes originated at the prokariotic level whereas the mitochondrial solute carriers must have developed when the ancestor prokaryote became symbiotic in the eukaryotic cell as they fulfill a new demand of the eukaryote for intensive traffic of metabolites between the cytosolic and matrix space (6

The breakdown of fatty acids, performed by the ␤-oxidation cycle, is crucial for plant germination and sustainability. ␤-Oxidation involves four enzymatic reactions. The final step, in which a two-carbon unit is cleaved from the fatty acid, is performed by a 3-ketoacyl-CoA thiolase (KAT). The shortened fatty acid may then pass through the cycle again (until reaching acetoacetyl-CoA) or be directed to a different cellular function. Crystal structures of KAT from Arabidopsis thaliana and Helianthus annuus have been solved to 1.5 and 1.8 Å resolution, respectively. Their dimeric structures are very similar and exhibit a typical thiolase-like fold; dimer formation and active site conformation appear in an open, active, reduced state. Using an interdisciplinary approach, we confirmed the potential of plant KATs to be regulated by the redox environment in the peroxisome within a physiological range. In addition, co-immunoprecipitation studies suggest an interaction between KAT and the multifunctional protein that is responsible for the preceding two steps in ␤-oxidation, which would allow a route for substrate channeling. We suggest a model for this complex based on the bacterial system.Fatty acids are fundamental biomolecules that are abundant in all life forms. With their enormous variation in chain length and degree of saturation, they are essential for energy storage, form structural entities in biomembranes, and serve as signaling molecules. Fatty acids are broken down in a cyclic manner, two carbons at a time, to generate a range of products by the process known as ␤-oxidation (1). In higher plants (2) and yeast (3) ␤-oxidation of all forms of fatty acid occurs in the peroxisomes. In plants, ␤-oxidation is essential for a plethora of physiological roles including responses to senescence and starvation, fatty acid turnover, and the regulation of plant lipid composition. Germinating seeds depend on ␤-oxidation for the mobilization and release of energy stored in the seed (4). Arabidopsis thaliana seeds deficient in ␤-oxidation enzymes are unable to germinate without an external sugar source; they have large and unusual peroxisomes and accumulate C16 -C20 fatty acids (5, 6). ␤-Oxidation is also responsible for the synthesis of jasmonic acid (7) and indole-3-acetic acid (auxin) via conversion from indole-3-butyric acid (8), which serve as crucial plant hormones regulating plant development and responses to biotic and abiotic stress. Hydrogen peroxide, produced as a byproduct during ␤-oxidation, is used by catalase to oxidize different toxins (e.g. alcohols) and plays an important role in cellular signaling (9).␤-Oxidation comprises four reactions. First, the CoA-activated acyl chain is oxidized to 2-trans-enoyl-CoA by an acylCoA oxidase (10), producing H 2 O 2 as a byproduct. The double bond is then reduced by 2-trans-enoyl hydratase forming L-3-hydroxyacyl-CoA followed by oxidation by NAD ϩ -dependent L-3-hydroxyacyl-CoA dehydrogenase. Both the hydratase and dehydrogenase activities are performed by a multifunction...

Thiolase I and II coexist as part of the glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons, the only system shown to have both forms. The importance of thiolases can be underscored not only by their ubiquity, but also by their involvement in a wide variety of processes in plants, animals and bacteria. Here we describe the cloning, expression and purification of acetoacetyl CoA thiolase (AACT) in enzymatically active form. Use of the extensive amount of sequence information from the databases facilitated the efficient generation of the gene-specific primers used in the RACE protocols. The recombinant AACT (1233 bp) shares 75% similarity with other plant AACTs. Comparison of specific activity of this recombinant AACT to a previously reported enzyme purified from primary sunflower cotyledon tissue was very similar (263 nkat/mg protein vs 220 nkat/mg protein, respectively). Combining the most pure fractions from the affinity column, the enzyme was purified 88-fold with a 55% yield of the enzymatically active, 47 kDa AACT.