Endoplasmic Reticulum, Oleosins, and Oils in Seeds and Tapetum Cells

Diverse organisms contain neutral lipids in subcellular particles for food reserves and other purposes. These lipid particles are present in seeds, flowers, pollen and fruit of higher plants, the vegetative and reproductive organs of primitive plants, algae, fungi, nematodes, mammalian glands and brown adipose tissue of mammals, and bacteria. Of all these lipid particles, the oil bodies (OBs) in seeds are the most prominent and best studied. Seeds of most plant species store oils (triacylglycerols [TAGs]) as a food reserve for germination and postgerminative growth. TAGs are present in small subcellular spherical OBs of approximately 1 μm in diameter. Each OB has a matrix of TAGs surrounded by a layer of phospholipids (PLs) and structural proteins termed oleosins. The small size of OBs provides a large surface area per unit TAG, which would facilitate lipase binding and lipolysis during germination. OBs inside the cells of mature seeds or in isolated preparations are remarkably stable and do not aggregate or coalesce. This stability is in contrast to the instability of artificial liposomes made from amphipathic and neutral lipids; the liposomes gradually coalesce after formation. Seed OBs are stable because their surface is shielded by a layer of oleosins. In maturing seeds, TAGs, PLs, and oleosins are synthesized in the endoplasmic reticulum (ER), from which budding OBs are released. Research on seed OBs and oleosins has been reviewed by Huang (1992, and an earlier Update article in 1996), Napier et al. (1996), Galili et al. (1998), Frandsen et al. (2001), and Murphy (2001). This Update provides brief reviews of earlier work and emphasizes recent major discoveries. Detailed information and references to earlier work can be found in the previous reviews. Models of an oleosin molecule, a seed oil body, and the synthesis of an oil body on the endoplasmic reticulum. A, The three portions of an oleosin molecule (yellow), showing the N-terminal hydrophilic portion, the central hydrophobic hairpin (and residues at the turn, including the Pro knot of three Pro residues and one Ser residue), and the C-terminal hydrophilic portion. The number of residues and their ranges in the 3 portions in all 17 Arabidopsis oleosins are shown. B, An OB having oleosins (yellow) and PLs (red) enclosing the matrix TAGs (blue). All molecules are drawn approximately to scale, whereas the diameter of the OB has been reduced 24 times to magnify the surface structure. C, A budding OB being produced on the RER. The ER lumen, the two PL layers (red), the sequestered TAGs (blue) in a budding OB, a ribosome with an mRNA synthesizing an oleosin polypeptide (dark line, of an unknown configuration), and enzymes (irregular circles) for the synthesis of TAGs and PLs are shown. More than 200 genes encoding oleosins have been identified. Nonplant organisms do not have oleosins. Recently, genes encoding oleosins on the storage OBs in Arabidopsis pollen (Kim et al., 2002) and tropical cacao (Theobroma cacao) seeds (Guilloteau et al., 2003) have been described. These findings have negated a proposal of having an alternative subcellular mechanism for stabilizing the OBs in pollen and another proposal explaining the short life span of tropical seeds such as cacao because of unstable OBs. The transcript of a gene encoding an oleosin in the moss Physcomitrella can be found in an expressed sequence tag database; this is the most primitive plant known to contain oleosins. Whether algae contain oleosins is not known. Arabidopsis has 17 genes encoding oleosins: 9 (8 in tandem on chromosome 5) that are active in the tapetum cells, 5 active in seeds, and 3 active in both seeds and pollen (Kim et al., 2002). Minor proteins present in isolated OBs of some seeds have been termed caoleosin and steroleosin (Frandsen et al., 2001). They do not have a long hydrophobic sequence, although they have sequences similar to but less conserved than the Pro knot sequence in oleosins (to be described). Their mode of association with the OBs and the possibility of their being contaminants of the OB fractions need to be explored. An oleosin molecule can be divided into three portions according to its amino acid sequence (Fig. 1A). The N-terminal portion can be short or long (e.g. 6–68 residues in Arabidopsis) and is hydrophilic. The central portion is a long hydrophobic stretch of 72 residues. The C-terminal portion can be short or very long (e.g. 28–1,000 residues in Arabidopsis oleosins), and its approximately 30 residues adjacent to the central hydrophobic stretch can form an amphipathic α-helical structure that interacts horizontally with the charged phosphate and choline groups of the PL layer on the OB surface. The C-terminal portions of most Arabidopsis oleosins consist of fewer than 100 residues; a few have 100 to 150 residues, one has 403, and another has 1,000 residues. Each of the longer C-terminal portions contains many repeats of short peptides, which are not conserved among oleosins and may not have functional significance. Some of the repeated short peptides have several Gly residues; thus, these oleosins are Gly-rich. These proteins are sometimes called Gly-rich proteins, although such a term does not describe the important characteristics of the proteins. The central hydrophobic stretch of 72 uninterrupted hydrophobic residues is the hallmark of an oleosin. No other protein in any organism has such a long hydrophobic stretch. Proteins on the surface of extracellular or intracellular lipid droplets, such as apolipoproteins, perilipin, adipophilin, and caveolin in mammals, phasin in bacteria, and the plastid lipid-associated protein (PAP), do not have a long hydrophobic stretch; their polypeptides run parallel to the surface of, rather than penetrate into, the lipid droplets. The 72-residue hydrophobic stretch of an oleosin is long enough (a trans-membrane [PL bilayer] peptide has 20–25 residues) to form a hairpin that penetrates the surface PL monolayer of an OB into the matrix (Fig. 1B). The center of the hydrophobic stretch has three Pro residues and one Ser residue that could interact to form a “Pro knot.” This proposal (Fig. 1A) is based on the presence of the relatively less hydrophobic Pro and Ser residues among the other more hydrophobic residues and TAGs in the matrix of an OB, as well as the well-known fact that Pro residues on polypeptides are breakers or turners of α-helical and β-structures. The formation of the Pro knot could permit the creation of a hairpin structure of the whole hydrophobic stretch, with 2 arms of 30 residues each joined by a turn of 12 residues. The 72 residues of the hydrophobic stretch are conserved in terms of hydrophobicity among oleosins of diverse species, and the conservation is much higher at the Pro knot and its immediate vicinity (-PX5SPX3P-). All oleosins contain the three Pro and one Ser residues at identical locations in the center of the hydrophobic stretch. All researchers agree with the central hydrophobic stretch forming a hairpin structure with a Pro knot at the turn but disagree on the secondary structures of the two hairpin arms. Earlier, the two arms were proposed to be an anti-parallel α-helical structure on the basis of an algorithm prediction (actually no database for predicting secondary structures of polypeptides in a hydrophobic environment exists) or an anti-parallel β-structure on the basis of high symmetry of residues between the two arms (Huang, 1996). If the two arms had an anti-parallel β-structure, they could bend at several locations where pairing of small Gly residues occurs; the bending would create more interactions among residues and thus offer higher stability. Two laboratories used circular dichromism and Fourier transform infrared spectroscopy to determine the secondary structures of the oleosin hairpin in its imitated native conditions; they came to opposite conclusions of an α-helical structure (Alexander et al., 2002) or β-structure (Li et al., 2002). The controversy underlines the difficulties in measuring the uniquely long hydrophobic polypeptide in a neutral-lipid environment. In addition, the arms or even the turn could interact with those in adjacent oleosins in the OB matrix. Such interactions could provide higher stability to the oleosin hairpin, in which the peptide bonds are relatively hydrophilic, in the hydrophobic environment. In maize, oleosins of two isoforms coexist in a 1:1 ratio, and interaction between the pair is likely. Furthermore, the N- and C-terminal portions of an oleosin, even though on the OB surface, may play a role in maintaining the hairpin in a special configuration. It has been hypothesized that the long hydrophobic stretch was evolved from duplications of a trans-membrane peptide in a primitive plant or algae. This could explain the residue symmetry of the 2 arms of the hairpin structure and the length of the 72-residue hydrophobic stretch being 4 times that of a trans-membrane peptide (2 × 2 × approximately 20 residues; Huang, 1996). The hypothesis can be tested by comparing the amino acid sequences of oleosins (and the nucleotide sequences of the genes) with those of transmembrane segments of proteins, especially of enzymes related to TAG synthesis, in the most primitive organisms (currently, the moss Physcomitrella). Other than the hairpin hydrophobic stretch, the N- and C-terminal portions are quite variable, which indicates minimal constraints on their structures to perform functions. Analyses of the nine oleosin genes, all but one in tandem, in Arabidopsis chromosome 5 have confirmed the rapid evolution of the N and C termini of tapetum oleosins (Fiebig et al., 2004; Schein et al., 2004), as has been observed in seed oleosins. It is likely that for oleosins to perform their functions, the major structural requirement is the central hydrophobic stretch. The N- and C-terminal portions of an oleosin on the surface of a seed OB may act as a receptor for the binding of lipase or glyoxysomes during germination. This possibility can be tested. The test can be made with use of seed lipase protein derived from a cloned lipase gene or with Arabidopsis mutants defective of glyoxysomal surface proteins. OBs and their constituent TAGs, PLs, and oleosins are all synthesized on the ER. Diacylglycerol acyltransferase (DAG AT), the last enzyme and the only one unique to the synthesis of TAG, as well as enzymes for the synthesis of precursor DAG and PLs, are associated with the rough ER (RER). It is most likely that an alternative TAG-synthesizing enzyme that can transfer the acyl moiety from PLs instead of acyl-CoA to DAG is also in the ER. The presence of these enzymes in the ER is not in of the hydrophobicity of the TAG and its TAGs synthesized in the ER are sequestered in the hydrophobic the acyl of the PL of TAG at a of the ER a budding OB, which is by a layer of PLs (Fig. This budding OB with a PL monolayer is by the of oleosins to its surface. The with a oleosin peptide can be to the ER the The mRNA for the synthesis of oleosin is associated with the RER. of oleosin mRNA in an in synthesis is or or are and et al., et al., and 2002). The findings that of the oleosin mRNA after binding of the to the peptide and the synthesized oleosins are into the ER. In addition, stable of the in oleosin in synthesis contain into is the receptor on the is with and this can be with with an oleosin gene and the oleosin to the OBs et al., the are mutants defective in the oleosin is not to the and the oleosin is et al., The of oleosin to the ER with use of and from mammals, or plants, and thus the unique of the is the in the oleosin oleosins produced gene can be tested for their stable into in or the ER in et al., and 2002). The N- and C-terminal portions of the oleosin molecule are relatively in the protein to the ER. the long hydrophobic stretch of the oleosin is the for No sequence in the hydrophobic stretch is any of the and sequences the hydrophobic stretch can the protein to the ER. the conserved Pro knot is not because of the three Pro residues with residues does not of the oleosin to the The that peptides the hydrophobic stretch can be the is with the that the hydrophobic of an can a diverse of hydrophobic peptides at the N termini or of many proteins. The oleosin polypeptide synthesized or being synthesized on the ER a on the basis of its hydrophobic and hydrophilic interactions with the PL The hydrophilic N- and C-terminal portions interact with the PL layers on the of the ER (Fig. The central hydrophobic stretch in the hydrophobic acyl portion of the PL is much from in and in for such a of the oleosin et al., et al., The N- and C-terminal portions but not the hydrophobic stretch of the oleosin in isolated are to by this is similar to that of the oleosins on mature OBs. The secondary structure of the 72-residue hydrophobic stretch in the hydrophobic portion of the PL is unknown but likely from that in a mature The hydrophobic of the ER does not provide an of hydrophobic for the hydrophobic stretch to its most stable hairpin but the matrix of a mature OB The hydrophobic stretch of the oleosin the hydrophobic of the ER could a hairpin structure or an structure with or parallel to the PL (Fig. An is the of the hydrophobic of the PL the ER is synthesizing also of TAGs, which be sequestered in and thus the hydrophobic of the PL the hydrophobic of the PL may have more for the hydrophobic stretch of an oleosin than that by the length of the two acyl the synthesized oleosins and the TAGs on the ER to the budding OBs. This is made in with the of and The TAGs and the oleosins both be more stable in the hydrophobic environment of a budding A native oleosin into the ER to the budding OB, but a oleosin may The has been used to the on the oleosin that the protein on the ER to to the OB et al., 2004; and 2002). The mechanism of this oleosin has been by in with use of oleosins and of oleosins in the ER and OB this not per but also the stability of the oleosins in OBs. oleosins that can to the OBs may be unstable and be by native The for an oleosin to to and be into the OBs are similar to those for the protein to the more are The Pro knot in the hydrophobic stretch is also for the stable into the OBs. It is likely that a oleosin the Pro knot (e.g. having the three Pro residues with can into the ER and also to the OB but is unstable and thus by native In to the need for the Pro length or of the N- or C-terminal portions or length of the hydrophobic stretch to a reduced of the oleosin in OBs. be on the of the ER to be to to the budding OBs. An N-terminal ER peptide from a protein to the N of an oleosin, produced gene can the N-terminal portion of the oleosin but not the hydrophobic stretch with or the C-terminal portion into the ER et al., the hydrophobic interaction between the long hydrophobic stretch and the acyl of the PL with or the hydrophilic interaction between the C-terminal portion of the oleosin and the PL layer on the is for the oleosin to the PL into the This oleosin can be into the ER but be into the OBs. its polypeptide the whole PL of the ER to the PL monolayer of a budding OB (Fig. would be unstable in the OBs. a be to the ER or budding OBs the of the All the from in that the can be If the is not in the with the hydrophobic stretch in could to the hydrophobic of has been that in oleosin into mature OBs or et al., a mature OB is with oleosins on its surface and has no for oleosins. In oleosin can into artificial OBs surface has not been with oleosins and 2001). A with the hydrophobic stretch could to the hydrophobic of the ER or a budding OB surface has not been with oleosins. the for the requirement for the to the oleosin to the ER has from in with mutants defective in et al., This with be tested with In addition, oleosin synthesis both the and a mechanism has not been the synthesized TAGs and oleosins on the ER to and at the budding OB, a of of these two from the of synthesis to the budding This can explain the that more oleosins are present in the ER the budding OBs Whether of ER for TAG and oleosin synthesis are present to be In an in seed with synthesized TAGs, and after this synthesis, were by into fractions et al., The with the more TAG, oleosin, and lipid synthesis on a per This may ER for TAG and oleosin synthesis, or of ER to the budding OBs and thus have more TAGs and a In an earlier an of maturing was by DAG the last and unique enzyme for TAG synthesis, was found with in of diverse and Huang, the DAG was not in ER with the and most TAGs fewer in the cells, TAGs are likely synthesized in diverse of the ER and to the budding OBs. a budding OB on the ER be (Fig. An a OB, and The size of an OB is or by the or of synthesis of oils and oleosins. a high by have whereas have OBs with surface. If the cells do not as those in the of the OBs very large a special mechanism for the of the budding OB from the The oleosins on the surface may interact among to a of at the of the the the may proteins (e.g. or of the ER or This possibility can be tested by for Arabidopsis mutants seeds have or OBs or only budding OBs with use of after lipid some of these mutants may be defective in the for the of OBs from the ER. In the of such as oil and each has only several large lipid which the of the is or no oleosins on the lipid The lipids are for to for seed and thus are not to be in small as the OBs in the TAGs are synthesized in the as are the seed but the of oleosins (Fig. a the budding OB only by PL is from the this is to the synthesis of OBs in having a high It is that the cells can be to small OBs instead of large lipid oleosin is to be this can be in is because the oil and are The presence of oleosins in tapetum cells of in Arabidopsis and was a from gene et al., et al., because tapetum cells were not known to contain OBs similar to those in These oleosins are present in a neutral which has been termed the because of its unique presence in tapetum the findings of oleosins in tapetum cells are to the especially and In nine genes the tapetum of which are in tandem on chromosome 5 (Kim et al., et al., 2004; Schein et al., of these genes is expressed to an oleosin of which of all the tapetum oleosins. of the other Arabidopsis tapetum oleosins are but one has has a similar oleosin gene et al., and et al., and the most active gene a major oleosin of or the or encoding the tapetum oleosins have rapid likely because the constraints for protein structures and thus are not oleosins are in a called the et al., The tapetum is a layer enclosing the in which mature to cells are the only cells that are very active and the of the an of the tapetum cells are for active and contain and a of the cells have a of to be the maturing pollen as the pollen In the tapetum cells at this of are with two storage the and the et al., The of 3 to 4 μm in are of but with small spherical lipid of by the structural protein of similar can be found in cells, such as fruit and cells, the are unique to the tapetum Each spherical of 2 to 3 μm in has of TAGs, and by oleosins and PLs as those in seed oil and derived from the ER. The of the and are and to the after the of the tapetum cells during the of The but not the TAGs of the and the but not the structural protein of the are and to the pollen surface, forming the of the pollen et al., The and mechanism for the are It is that in seed TAGs are the for and the oleosins are the whereas in the oleosins may be the for (to be and TAGs are the The tapetum TAGs after the of the do they They could be used as an for active of the tapetum Their could be used to as their could be to as one of the two major lipid other being the for the maturing These are with Arabidopsis mutants defective in tapetum TAG synthesis or the and other lipids on the pollen form a the of the oleosins is not In the or oleosin on the pollen has been into two one the N-terminal portion and the central hydrophobic stretch, and the other the long hydrophilic C-terminal portion and et al., Whether other oleosins on the pollen are is not known. The may not have as be described. The most oleosin on the pollen has a large size in Arabidopsis and or in to its of repeats of short peptides at its C Each of these repeats several Gly residues, which the protein Gly-rich. of its Gly-rich this oleosin (and to other on the pollen has been to be in with the of the Such a be with have rapid and both the tapetum and seed oleosins have repeats of short peptides at the C some of these repeats have high Gly whereas do The and of the at the C termini may the minimal structural constraints on this of the protein to perform functions. The high Gly at the C termini of oleosins may be and the Gly-rich C termini in some seed oleosins do not have an for interaction with the In the short repeats at the C termini of the most tapetum oleosins have not high Gly but also high Ser and the oleosin also and Arabidopsis oleosin has and and the oleosin and and An oleosin molecule may on the pollen and on the because of its amphipathic N- and C-terminal portions are hydrophilic, and its central portion is The amphipathic oleosin can act as an to the pollen with and other It can also for germination after the pollen has on the have and be drawn from the to the pollen for germination and growth. and other neutral lipids are not and no other is known to be to act as a The and amphipathic oleosins could be such a the basis of these two proposed functions, the of repeats of short peptides, which are all hydrophilic, to the C termini of oleosins and the of the or oleosins into two do not the of the oleosins. The proposed are in with the that the pollen of an Arabidopsis in the major pollen oleosin does not on the and The major structural constraints on the oleosins to perform the proposed are a long hydrophobic stretch to interact with the TAG in the a per but for storage in the and an amphipathic molecule to the pollen and from the All the observed rapid on the tapetum oleosins have not these A of the synthesis of a in tapetum A, of an oil from the by a mechanism similar to that in Each oil of an oil matrix (blue) by layers of PL (red) and oleosins of PLs and oleosins on the oil is B, of several oil and ER C, A maturing ER A mature from et al. is the of the in the The of their and in with those of the ER are not this information is These in the may the transfer of oleosins from the tapetum cells to the pollen surface. They may proteins, such as and other proteins, for the pollen these proteins would on the They may contain such as and for the pollen these would the structures of the they may contain and other secondary for the pollen these are well-known but of functions. and can be used to test the presence of these in the and the interaction between the ER and the during of the tapetum oleosins and at the findings to species, the structure and between the and the the of the oleosins on and the of the in the and are In addition, use of Arabidopsis mutants defective in these was by in the