B

Oleosin 2012

Fig. 3.5 A proposed scheme for the formation of oil bodies. Oil bodies probably bud off from the ER as small TAG droplets of 60-100 nm diameter. Such small oil droplets are unstable in aqueous media and would tend to coalesce, rapidly at first and then more slowly until reaching a stable size. (A) In the absence of oleosins and in non-desiccating tissues like palm mesocarp, the final oil body size is about 10-25 p.m. (B) Directly after their synthesis, oleosins are inserted into the ER membrane, from where they may diffuse into nascent oil bodies, probably undergoing a conformational change. Alternatively, oleosin-poor oil bodies may re-fuse with ER to undergo TAG turnover and acquire more oleosins. If the ratio of oleosin:TAG synthesis is only low to moderate, the nascent oil bodies will need to undergo many fusion events before acquiring a complete oleosin monolayer. In oilseed that has oil bodies of 1-4 p.m, approximately 9-14 fusions will be required to get from a diameter of 100 nm to 1-4 p.m. (C) With much higher ratios of oleosin: TAG synthesis, fewer fusions are required before the oleosin monolayer is complete, which results in smaller oil bodies. Therefore in rapeseed, mature oil body sizes of 0.2-1.0 p.m can be achieved by only 3-8 fusion events from 100 nm oil bodies. Reprinted from Fig. 1 in D.J. Murphy, C. Sarmiento, J.H.E. Ross & E. Herman, Oleosins: their subcellular targeting and role in oil-body ontogeny, in Physiology, Biochemistry and Molecular Biology of Plant Lipids (eds J.P. Williams, M.U. Khan & N.W. Lem) with kind permission of Kluwer Academic Publishers.

Fig. 3.5 A proposed scheme for the formation of oil bodies. Oil bodies probably bud off from the ER as small TAG droplets of 60-100 nm diameter. Such small oil droplets are unstable in aqueous media and would tend to coalesce, rapidly at first and then more slowly until reaching a stable size. (A) In the absence of oleosins and in non-desiccating tissues like palm mesocarp, the final oil body size is about 10-25 p.m. (B) Directly after their synthesis, oleosins are inserted into the ER membrane, from where they may diffuse into nascent oil bodies, probably undergoing a conformational change. Alternatively, oleosin-poor oil bodies may re-fuse with ER to undergo TAG turnover and acquire more oleosins. If the ratio of oleosin:TAG synthesis is only low to moderate, the nascent oil bodies will need to undergo many fusion events before acquiring a complete oleosin monolayer. In oilseed that has oil bodies of 1-4 p.m, approximately 9-14 fusions will be required to get from a diameter of 100 nm to 1-4 p.m. (C) With much higher ratios of oleosin: TAG synthesis, fewer fusions are required before the oleosin monolayer is complete, which results in smaller oil bodies. Therefore in rapeseed, mature oil body sizes of 0.2-1.0 p.m can be achieved by only 3-8 fusion events from 100 nm oil bodies. Reprinted from Fig. 1 in D.J. Murphy, C. Sarmiento, J.H.E. Ross & E. Herman, Oleosins: their subcellular targeting and role in oil-body ontogeny, in Physiology, Biochemistry and Molecular Biology of Plant Lipids (eds J.P. Williams, M.U. Khan & N.W. Lem) with kind permission of Kluwer Academic Publishers.

There is an important difference between animals and plants with respect to TAG composition and metabolism. Plants must of necessity synthesize their TAG from simple starting materials according to their requirements, since they have no dietary source of preformed lipids. Unlike animals, they have the ability to synthesize the linoleic acid that they normally possess in abundance: animals rely on plants for this essential nutrient (Section 4.2.3). The fatty acid composition of animal TAG is greatly influenced by their diet and, therefore, ultimately by the plant materials they eat. The way in which dietary TAG are modified by animals may differ between species and from organ to organ within a species. Such modifications are not necessarily always effected by the animal's own cells. For example, in ruminants such as cows, sheep or goats, the micro-organisms present in the rumen hydrogenate the double bonds of dietary polyunsaturated fatty acids like linoleic and a-linolenic acids to form a mixture of mainly saturated and cis and trans monoenoic acids (Section 2.2.5). An outstanding and significant feature of TAG fatty acid composition is that it is quite distinctly different from that of the phospholipids or the non-esterified fatty acid (NEFA) pool. An understanding of this cannot be obtained simply from analyses of fatty acid distributions but must depend upon the study of different metabolic pathways, and of the individual enzymes in those pathways that are involved in the biosynthesis of each lipid class.

3.4.1 Pathways for complete (tfe novo) synthesis build up TAG from small basic components

3.4.1.1 The glycerol 3-phosphate pathway in mammalian tissues provides a link between TAG and phospholipid metabolism

Historically, the de novo pathway, now usually known as the glycerol phosphate pathway for TAG biosynthesis (Fig. 3.6), was worked out first. It was first proposed by the American biochemist, Kennedy, based on the earlier work of Kornberg and Pricer, who first studied reactions 1 and 4 (Fig. 3.6), the formation of phosphatidic acid by stepwise acylation of glycerol 3-phosphate.

Kennedy also demonstrated the central role of phosphatidic acid in both phospholipid (Section 7.1.2) and TAG biosynthesis and one of his outstanding contributions was to point out that diacyl-glycerols derived from phosphatidic acid form the basic building blocks for triacylglycerols as well as phosphoglycerides.

It is now known that steps 1 and 4 in Fig. 3.6, the stepwise transfer of acyl groups from acyl-coen-

zyme A to glycerol 3-phosphate, are catalysed by two distinct enzymes specific for positions 1 and 2. The enzyme that transfers acyl groups to position 1 (acyl-CoA:glycerol phosphate 1-O-acyltransferase, GPAT) exhibits marked specificity for saturated acyl-CoA thiolesters whereas the second enzyme (acyl-CoA:1-acyl glycerol phosphate 2-O-acyl-transferase, sometimes referred to as lysophos-phatidate acyltransferase, LPAT) shows specificity towards mono- and dienoic fatty acyl-CoA thiol-esters. This is in accord with the observed tendency for saturated fatty acids to be found in position 1 and unsaturated ones in position 2 in the lipids of most animal tissues.

GPAT, which transfers an acyl group to position 1, has been cloned and sequenced. There are two isoforms in mammals, one associated with the endoplasmic reticulum, the other with the outer mitochondrial membrane. The active sites of both isoforms face the cytosol. In the liver of most mammals studied so far, there is a similar activity at both subcellular locations, whereas in other tissues, the microsomal enzyme has about ten times the activity of the mitochondrial isoform. It is not yet known whether the two isoforms have different functions but there is experimental evidence to link changes in activity of both enzymes with changes in overall TAG biosynthesis. For example, when cultured 3T3-L1 cells differentiate into mature adipo-cytes, the activity of microsomal GPAT increases about 70-fold and the mitochondrial activity (and the amount of its mRNA) increases about tenfold. It is interesting to speculate whether these questions might be resolved by manipulating 3T3-L1 cells to produce lines in which the mitochondrial GPAT gene is either deleted or over-expressed.

GPAT was first purified from E. coli membranes and more recently a rat liver mitochondrial enzyme has also been purified. The purified E. coli enzyme, with a molecular mass of 83kDa, proved to be inactive, but activity was restored by reconstitution with phospholipid preparations, principally cardi-olipin and phosphatidylglycerol. A 90 kDa protein ('p90'), which has about 30% identity with the E. coli GPAT, has been cloned in mice. When the mice were fasted and then given a diet rich in carbohy-

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