Synthesis of Oils Oleosins and Oil Bodies in Endoplasmic Reticulum

Oil bodies, including their constituent TAGs, PLs, and oleosins, are synthesized on ER. Diacylglycerol acyltransferase (DAG AT), the last enzyme and the only one unique to the synthesis of TAGs, as well as enzymes for the synthesis of precursor DAGs and PLs, are associated with rough ER (RER). An alternative TAG-synthesizing enzyme, which can transfer the acyl moiety from PLs instead of acyl-CoA to DAG, is also located in ER. The presence of these enzymes in ER is not surprising in view of the hydrophobicity of TAG and its metabolic precursors. TAGs synthesized in ER are sequestered in the hydrophobic region (i.e., the acyl region of the PL bilayer). Continuation of TAG accumulation at a domain of ER forms a budding OB, which is enclosed by a single layer of PLs (Fig. 1c). This budding OB, covered with a PL monolayer, is stabilized by inclusion of oleosins to its surface.

Ribosome-mRNA with a nascent oleosin peptide can be guided to ER via the signal-recognition particle (SRP) pathway. mRNA for synthesis of oleosin is associated with RER. Translation of oleosin mRNA in an in vitro synthesis system is retarded or enhanced, respectively, when SRP or microsomes are added (Abell et al. 2002; Beaudoin and Napier 2002; Loer and Herman 1993; Thoyts et al. 1995). The findings suggest that the translation of oleosin mRNA pauses after binding of SRP to the nascent peptide and accelerates when the newly synthesized oleosins incorporate into ER. In addition, stable incorporation of in vitro-synthesized oleosin (commercial in vitro synthesis systems usually contain SRP) into microsomes is inhibited when the SRP receptor (SRP-60) on microsomes is removed beforehand through proteolysis. This inhibition can be restored with reconstituted SRP receptor. Yeast transformed with an oleosin gene synthesizes and targets oleosin to OBs (Ting et al. 1997). When the transformed yeast strains are mutants defective in SRP components, oleosin is not targeted to OBs, and the nontargeted oleosin is proteolytically degraded (Beaudoin et al. 2000).

Targeting of oleosin to ER in vitro can occur with the use of SRP components and microsomes from yeast, mammals, or plants. Thus the unique aspect of the targeting mechanism is the targeting signal(s) in the oleosin molecule. Modified oleosins produced via gene recombination can be tested for their stable insertion into microsomes in vitro or into ER in vivo (Abell et al. 1997, 2002; Beaudoin and Napier 2002). The N- and C-terminal portions are relatively unimportant in targeting oleosin to ER. Rather, the long hydrophobic stretch of oleosin is the predominant factor for targeting. No specific signal sequence in the hydrophobic stretch is required. Instead, any of the multiple and probably overlapping sequences along the hydrophobic stretch can target the protein to ER. Significantly, the highly conserved proline knot is not important for the in vitro targeting of oleosin to microsomes, because replacement of the three proline residues with leucine residues does not affect the targeting. The finding that multiple peptides along the hydrophobic stretch can be the targeting signals is consistent with the knowledge that the hydrophobic pocket of an SRP can recognize a diverse array of hydrophobic ER-targeting peptides at the N termini or interior of many proteins.

The nascent oleosin polypeptide synthesized or being synthesized on ER assumes a topology on the basis of its hydrophobic and hydrophilic interactions with the PL bilayer. The hydrophilic/amphipathic N- and C-terminal portions interact with the PL layers on the cytosolic side of ER (Fig. 1c), whereby the central hydrophobic stretch buries itself in the hydrophobic acyl portion of the PL bilayer. Much evidence from in vivo and in vitro experiments exists for such a topology for the nascent oleosin (Abell et al. 1997, 2002; Beaudoin et al. 2002). The N- and C-terminal portions, but not the hydrophobic stretch, of oleosin in isolated microsomes are susceptible to pro-teolysis by exogenously added proteases; this observation is similar to that of oleosins on mature OBs. The secondary structure of the 72-residue hydrophobic stretch in the hydrophobic portion of the PL bilayer is unknown but likely differs from that in a mature OB. The matrix of a mature OB, but not the hy-

drophobic region of ER, provides an excess of hydrophobic volume for the hydrophobic stretch to assume its presumably most stable hairpin configuration. The hydrophobic stretch of oleosin within the hydrophobic region of ER could assume a bended hairpin structure or an extended structure with or without coiling, running parallel to the PL bilayer (Fig. 1c). An additional consideration is the actual thickness of the hydrophobic region of the PL bilayer. While ER is synthesizing oleosins, it also produces massive amounts of TAGs, which will be temporarily sequestered in, and thus enlarge, the hy-drophobic region of the PL bilayer. Thus, the hydrophobic region of the PL bilayer may have more room for the hydrophobic stretch of a nascent oleosin than that defined by the length of the two acyl chains.

Both the newly synthesized oleosins and the temporarily located TAGs on ER diffuse to budding OBs. This movement is made possible in accordance with the fluid mosaic model of membrane action and thermodynamic considerations. TAGs and the oleosins will be more stable in the hydrophobic environment of a budding OB. A native oleosin stably inserted into ER diffuses to the budding OB, but a stably inserted, artificially modified oleosin may not. The mechanism of this oleosin movement has been studied in vivo through using modified oleosins and measurements of oleosins recovered in ER and OB fractions (Abell et al. 1997, 2002, 2004; Beaudoin and Napier 2002). Strictly speaking, this approach measures not just targeting success per se, but also the stability of the modified oleosins in OBs. Modified oleosins that can diffuse to the budding OBs may be unstable there and removed by endogenous proteolysis. The molecular requirements for oleosin to diffuse successfully to, and incorporate stably into, OBs are similar to those for targeting the protein to ER; however, more are required. The proline knot in the hydrophobic stretch is also essential, presumably for stable anchoring of oleosin on OBs. A modified oleosin without the proline knot (e.g., having the three proline residues replaced with leucine residues) can probably insert into ER and also diffuse to the budding OB but would be unstable there and thus eliminated by endogenous proteolysis. In addition to the need for the proline knot, decreased length or elimination of the N- or C-terminal portions or decreased length of the hydrophobic stretch all lead to a reduced recovery of the modified oleosin in OBs.

Oleosins must be only on the cytosolic side of ER to be able to diffuse to the budding OB. Attempts to insert the whole oleosin molecule into the luminal side of ER have been unsuccessful. An N-terminal ER targeting peptide from a nonoleosin protein attached to the N terminus of an oleosin, produced via gene recombination, can pull the N-terminal portion of the oleosin but not the hydrophobic stretch (with or without the C-terminal portion) into the ER lumen (Abell et al. 2002, 2004). Apparently, the hydrophobic interaction between the long hydrophobic stretch and the acyl moieties of the PL bilayer (with or without the added hydrophilic interaction between the C-terminal portion and the PL layer on the cytosolic side) is too strong for the oleosin to leave the PL bilayer and insert into the lumen. This modified oleosin can incorporate into ER but cannot insert into the budding OB. Obviously, its polypeptide spanning across the whole PL bilayer of ER cannot diffuse to the PL monolayer of a budding OB (Fig. 1c). Even if it could, it would be unstable there.

It is uncertain whether a ribosome-mRNA-oleosin complex can target to ER or the budding OB directly without involvement of the SRP pathway. All the evidence from in vitro experiments shows that the SRP system is involved. A ribosome-mRNA-oleosin complex with the hydrophobic stretch dangling outward in vitro could bind to the hydrophobic pocket of an added SRP, regardless of whether SRP is actually involved in vivo. Certainly, it has been shown that oleosin synthesized in vitro cannot insert into mature OBs co- or posttranslationally (Hills et al. 1993). However, a mature OB is packed with oleosins on its surface and has no extra room for new oleosins. Oleosin synthesized in vitro can insert into artificial OBs whose surface has not been filled completely with oleosins (Chen and Tzen 2001). A ribosome-mRNA-oleosin complex with the hydrophobic stretch dangling outward could theoretically bind to the hydrophobic region of ER or a budding OB whose surface had not been filled completely with oleosins. Nevertheless, the strongest evidence for the need of SRP to guide oleosin to ER has come from in vivo studies with yeast mutants defective in SRP components (Beaudoin et al. 2000). This finding with yeast should be tested with plants. Further, whether oleosin synthesis employs both the SRP system and a direct insertion mechanism has not been evaluated.

As newly synthesized TAGs and oleosins on ER diffuse to and converge at the budding OB, a gradient of enrichment of these two components should exist from the point ofsynthesis to the budding OB. This concentration gradient can explain the immunocytochemical observation that more oleosins are present in the ER near the budding OBs (Herman 1987). Whether subdomains of ER for TAG and oleosin synthesis are present remains to be documented. In an in vitro study, sunflower seed microsomes supplied with precursors synthesized TAGs and, after this synthesis, were subfractionated by density gradient centrifuga-tion (Lacey et al. 1999). The fraction with the lowest buoyant density contained more TAG, oleosin, and lipid synthesis activity on a per fraction basis. This fraction may represent ER subdomains specialized for TAG and oleosin synthesis, or simply fragments of ER regions originally closest to the budding OBs and thus having more TAGs and a lower buoyant density. In an earlier experiment, when an extract of maturing maize kernel was subfractionated by density gradient centrifugation, DAG AT, the last and unique enzyme for TAG synthesis, was found with cytochrome reductase in RER fragments of diverse buoyant densities (Cao and Huang 1986). The DAG AT was not concentrated in ER fragments with the lowest buoyant densities and therefore most TAGs (or fewest polysomes). Thus, in the maize cells, TAGs are probably synthesized in diverse regions of ER and diffuse to the budding OBs. In the tapetum in

Brassica anthers, oleosin-coated oil droplets are structural analogs of seed oil bodies (see Sect. 4). During synthesis of these tapetum oil droplets, oleosin and the ER chaperone calreticulin were colocalized in extensive regions of the ER network, as seen in situ by immunofluorescence microscopy. Thus, the tapetum oleosins, and perhaps TAGs also, are synthesized in diverse regions of ER rather than in highly restricted ER subdomains.

A budding OB is released from ER as a solitary oil body (Fig. 1c). An early release will generate a smaller OB, and vice versa. The size and shape of an OB are determined in part or completely by the relative amount or rate of synthesis of oils and oleosins. High-oil maize kernels (having a high oil-to-oleosin ratio) generated by breeding have large, spherical OBs, whereas low-oil kernels have small OBs with irregularly shaped surface (Ting et al. 1996). In cells that do not synthesize oleosins, such as those in the fatty mesocarp of fruits, the OBs (lipid globules) become very large (see next paragraph). A special mechanism may exist for the physical release of a budding OB from ER. Oleosins accumulated on the bud surface may interact among themselves to produce a physical force of constriction at the neck of the bud, thereby releasing the OB. Or, the physical release may require specific cytosolic proteins (e.g., dynamins). These possibilities can be tested by screening for Arabidop-sis mutants whose seeds have larger or smaller OBs or only budding OBs viewed with a light microscope after lipid staining. Some of these mutants may be defective in the mechanism for physical release of OBs from ER.

In the fatty mesocarp of fruits such as avocado, oil palm, and olive, each cell has one to several large lipid globules, which occupy the bulk of the cell volume. Little or no oleosins are present on these lipid globules. Mesocarp lipids are for attracting animals and serve for seed dispersion and thus are not required to be in small entities such as seed OBs. Mostly likely, TAGs are synthesized in ER, as in seeds, but without a cosynthesis of oleosins (Fig. 1c). As a consequence, the budding OB enclosed only by PLs becomes larger (and/or fuses with adjacent budding OBs) before it is released from ER. This is equivalent to the synthesis of larger OBs in maize kernels having a high oil-to-oleosin ratio. It is possible that the mesocarp cells can be modified to synthesize small OBs instead of large lipid globules if oleosin is allowed to be cosynthesized with TAGs via genetic engineering. Although in theory this genetic engineering project can be easily achieved, in practice it is difficult because the better-known avocado, oil palm, and olive that contain fatty mesocarp are tree crops.

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