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The biogenesis of the plant seed oil body: Oleosin protein is synthesised by ER-bound ribosomes
Plant Physiol. Biochem., 1999, 37 (6), 481−490
The biogenesis of the plant seed oil body: Oleosin protein is synthesised by ER-bound ribosomes Frédéric Beaudoin, Dominic J. Lacey, Johnathan A. Napier* IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton Bristol BS41 9AF, UK * Author to whom correspondence should be addressed (fax +44 1275 394281; e-mail [email protected])
(Received December 15, 1998; accepted March 29, 1999) Abstract — An in vitro system was developed to demonstrate that the oleosin protein is synthesised on bound ribosomes and co-translationally inserted into the endoplasmic reticulum (ER) membrane. Microsomes were isolated with their associated bound ribosomes and transcripts from the developing embryos of sunflower (Helianthus annuus L.), and used to programme a cell-free translation system. The presence of the oleosin within the population of polypeptides synthesised by these rough membranes was demonstrated by immunoprecipitation with a specific oleosin antibody. Displacement of the bound ribosomes by the chelation of Mg2+ was also shown to displace the oleosin transcript. In vitro synthesised oleosin was shown to insert into sunflower microsomal membranes, with a portion of the protein being resistant to protease digestion. Radio-sequencing was used to determine the N-terminus of this fragment, providing new insights into the topology of the oleosin protein. © Elsevier, Paris Endoplasmic reticulum membrane / oleosin / oilseeds / seed development ER, endoplasmic reticulum / SRP, signal recognition particle / TAG, triacylglycerol
1. INTRODUCTION Oleosins are a unique class of proteins found exclusively in oil bodies of lipid-storing tissues such as seeds and pollen [10, 21, 22]. Oleosins are usually in the size range of 15–26 kDa, and have a characteristic three-domain structure. This takes the form of a highly conserved hydrophobic central domain of usually seventy residues, flanked by two amphipathic regions of a more variable nature. In most developing oilseeds, the oleosin protein is localised to the surface of the oil body (the site of triacylglycerol [TAG] deposition), and is postulated to play a role in both the biogenesis of the organelle [7, 8] and also in the prevention of aggregation of the storage lipid during seed maturation and desiccation [4]. Although the oil body is a relatively simple organelle, being made up of a TAG interior and an oleosin-phospholipid shell, the mechanisms by which it is formed are unclear and has been the subject of a degree of controversy [10, 21, 22]. What is clear is that storage lipid in the form of TAG is synthesised by the ER membrane [13, 27], probably between the leaflets of the membrane bilayer. This has led to speculation Plant Physiol. Biochem., 0981-9428/99/6/© Elsevier, Paris
that the oil body might arise as a result of concomitant synthesis of TAG and oleosin by the ER, with the oleosin being targeted and co-translationally inserted into the membrane by the signal recognition particle (SRP) pathway [22]. Although there is still some dispute as to the temporal regulation of TAG synthesis versus oleosin synthesis [4, 31], this SRP-dependent hypothesis has become widely accepted [1, 6]. This has been enhanced by studies on the targeting and deposition of the oleosin protein as a means of investigating oil body biogenesis. Initial studies by two independent groups indicated that in vitro synthesised oleosin was unable to insert into isolated oil bodies, but was able to insert into microsomal membrane extracts [9, 16]. Moreover, the in vitro synthesis of the oleosin was stimulated by the addition of microsomes, indicative of co-translational insertion. More recently, we have shown that this effect is due to the interaction between the oleosin and SRP [28] causing translational arrest which is relieved by the presence of the microsomal SRP receptor. However, all these in vitro studies made use of canine pancreatic microsomal membranes and in vitro transcribed (from cloned cDNA templates) mRNAs which, although a
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model system for the study of ER membrane protein targeting, is clearly non-homologous for developing oilseeds. Ultrastructural studies have been carried out to try and identify any association between the ER and the oil body and, whilst a number of authors have reported this phenomenon (see [8] for a review), others have failed to observe any such connections. Therefore, if the biogenesis of oil bodies is to be studied in detail, it is essential to develop novel methods. Stobart et al. [27] reported in vitro accumulation of TAG to high levels, using a microsomal membrane preparation derived from safflower cotyledons. The possibility of combining in vitro TAG synthesis with that of oleosin, resulting in in vitro synthesised oil bodies, might provide insights into organellar biogenesis [13, 22]. To facilitate the assembly of such a system, we decided to investigate the association of oleosin transcripts in vivo with membrane-bound ribosomes. Previously, Qu et al. [23] reported that the oleosin protein (or protein L3 as it was then designated) was synthesised by membrane-bound polysomes. This was based on their observation that cell-free in vitro translation products of RNA extracted from bound polysomes contained a polypeptide of similar mobility to that of the oleosin protein. This initial report has subsequently been cited as demonstrating the synthesis of oleosin on rough ER membranes (see, for example [1, 8, 9]) However, careful reading of the original data presented by Qu et al. [23] indicates that the identity of the putative rER-derived oleosin (L3) translation product was not confirmed immunologically, with identification based solely on electrophoretic mobility. Moreover, the abundance of this putative oleosin transcript among total ER-bound polyribosome mRNAs casts some uncertainty on its identity, since the data indicated that the translation product identified by Qu et al. [23] was the major, if not the only, protein synthesised by bound ribosomes. In contrast, other studies of a number of related species showed that among total protein expressed in developing seeds, the seed storage proteins are by far the most abundant class of polypeptide (see [26] for a review). It is also noteworthy that other workers have shown that oleosins are only minor components (< 5 %) of proteins immunoprecipitated from in vitro translated mRNA isolated from developing seeds [9]. These seed storage proteins are synthesised exclusively by ER-bound polyribosomes, prior to their entry into the plant secretory system [26]. These observations clearly indicate that further studies were required to verify the synthesis of oleosin by bound ribosomes on the rER. Therefore, we isoPlant Physiol. Biochem.
lated microsomal membranes from developing sunflower cotyledons with or without their associated ribosomes, based on the Mg2+ requirement for ribosome binding. These microsomes were then used to programme a wheat germ cell-free in vitro translation system, and resulting polypeptides were characterised by immunoprecipitation using a polyclonal antiserum raised against sunflower oil-bodies. This demonstrated that the sunflower oleosin transcripts are among the population of rER-associated mRNAs and that this protein is translated on bound ribosomes. Moreover, we also show that oleosin protein synthesised in vitro inserts into sunflower microsomal membranes in a carbonate-extraction-resistant manner, and proteaseprotection studies revealed polypeptides resistant to digestion of a similar size to those obtained with protease-digested sunflower-oil bodies.
2. RESULTS AND DISCUSSION 2.1. The isolation of transcripts associated with the rER of sunflower microsomes Previous studies of the insertion of oleosins into microsomal membranes have used canine pancreatic microsomes as a substrate [9, 16, 28]. We wished to carry out in vitro oleosin membrane insertion assays in a more homologous system, using not only plantderived microsomal membranes, but also the endogenous oleosin mRNAs associated with the polyribosomes bound to the membranes. Therefore, we isolated ER-derived microsomes from developing cotyledons of sunflower. Plant ER-derived microsomes can be isolated by sucrose density gradient centrifugation and separated into smooth- and rough-surfaced microsomes in presence of Mg2+ because of their different equilibrium densities [3, 17, 18, 33], the latter having a greater density as a result of the associated ribosomes. However, it has been demonstrated more recently that, in developing embryos of Brassica napus, the ER is heterogeneous with respect to its ability to synthesise storage lipid [12, 13] with the enzymes of TAG synthesis being associated with lighter membrane fractions with equilibrium densities ranging from 12 to 28 % sucrose (w/w), presumably due to their increased TAG content. Microsomes extracted from mid-maturation embryos of sunflower, in the presence or in the absence of EDTA, generated smooth and rough microsomes, respectively, and were added to a wheat germ cell-free extract to allow the in vitro translation of any endogenous mRNAs associated with the microsomal prepa-
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Figure 1. SDS-PAGE analysis of in vitro translated microsomes preparations and the polypeptide complement of associated rER-bound transcripts. A, Membranes were translated in a wheat germ system, the products separated by SDS-PAGE and radiolabelled proteins detected by autoradiography. Lane 1, Microsomes extracted in presence of 5 mM MgCl2 without EDTA; lane 2, microsomes extracted in presence of 1 mM MgCl2 and 5 mM EDTA; lane 3, wheat germ negative control incubated without membrane. B, Sunflower rough microsomes extracted in the presence of 5 mM MgCl2 (lanes 1, 3, 5) or in vitro transcribed preprolactin mRNAs (lanes 2, 4, 6) were translated using either: (a) normal wheat germ extract as positive control; (b) the 290 000 × g supernatant of wheat germ extract centrifuged for 1 h; (c) water added to the translation mixture instead of wheat germ as negative control. The products of the reactions were analysed as described previously. C, After in vitro translation, the rER membranes were resuspended and immunoprecipitated with either anti-oleosin antisera (lane 1) or pre-immune sera (lane 2) and analysed by SDS-PAGE and autoradiography. The positions of molecular mass markers is indicated.
rations. The radiolabelled translation products were analysed by SDS-PAGE and fluorography (figure 1 A). A negative control (no membrane added) was included to identify any endogenous wheat germ mRNA. After in vitro translation, the rough microsomes synthesised a number of proteins ranging from 15 to 90 kDa (figure 1 A, lane 1), whereas almost no labelled protein was detectable in the sample translated in the presence of smooth microsomes (figure 1 A, lane 2). This confirmed that in the presence of 5 mM EDTA, polysomes were released from the sunflower microsomes, and that the free cytosolic ribosomes were not pelleted with the membranes. The wheat germ control (figure 1 A, lane 3) gave a very faint band of 32 kDa, often observed with this in vitro translation system in the absence of exogenous template mRNAs. The bands obtained with the rough microsome preparation are the products of transcripts associated with the ribosomal population bound to these membranes (i.e. ribosome complexes which have been associated in vivo with the rER by virtue of a SRP-signal sequence present in the polypeptide nascent chain). The darkest bands correspond to the most abundant mRNAs associated with the microsomal-bound ribosomes, and probably represent seed storage proteins, such as 11S globulin subunits (helianthenins) and 2S albumins, which are highly transcribed in sunflower during mid-stage seed development [19, 29]. Storage proteins are well known
to be associated with, and synthesised on, membranebound polysomes, since they possess signal sequences to direct them into the ER lumen [6, 15]. However, transcripts encoding for oleosin are also synthesised at this stage of sunflower cotyledon development [28], and a number of polypeptides of similar mobility (∼20 kDa) to sunflower oleosins [32] are present. To confirm the rER-derived nature of the polypeptide profile, a membrane- and ribosome-free wheat germ extract was prepared by high speed centrifugation. The result obtained (figure 1 B) clearly demonstrated that whilst both the rER-associated transcripts, and control preprolactin mRNA translated efficiently in a normal wheat germ extract (figure 1 B, lanes 1, 2), only the microsome-associated polyribosomes (figure 1 B, lane 3), but not preprolactin (figure 1 B, lane 4), were translated in the centrifuged (i.e. cytosolic ribosomefree) wheat germ extract. The negative controls (figure 1 B, lanes 5, 6) did not show any background. This clearly shows that the polypeptide profile generated by our in vitro translation of sunflower rough microsomes was a result of the presence of transcripts associated in vivo with the rER.
2.2. Immunoprecipitation of oleosin from in vitro translated sunflower rER In contrast to the study of Qu et al. [23], a number of proteins were present in the in vitro translation vol. 37 (6) 1999
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products programmed by membrane-bound polysomal mRNAs indicating that a precise method was required to determine if the oleosin was indeed represented in this polypeptide complement. Therefore, the in vitro translation products of reactions programmed with rough (bound polysomes) sunflower microsomes (i.e. figure 1 B, lane 3) were immunoprecipitated with either a polyclonal antiserum to the sunflower oil body proteins [20] or pre-immune serum; this immunoprecipitation step was carried out after the microsomes and their associated translation products were purified through a sucrose-step gradient and carbonateextracted. The results obtained (figure 1 C) clearly show the immunoprecipitation of a z20-kDa polypeptide, the expected size of the sunflower oleosin (lane 1) [20, 32]. This band was not observed in the case of the pre-immune sera (lane 2). Thus, the sunflower oleosin was indeed synthesised by microsomal-bound ribosomes and, co-translationally inserted into the ER membrane in a carbonate-resistant manner [5]. The observation of oleosin insertion into plant microsomal membranes complements previous results obtained from in vitro insertion of oleosin into pancreatic canine microsomes [1, 9, 16, 28]. Interestingly, a recent study by Sarmiento et al. [24] demonstrated that oleosin protein was immunolocalised to regions of the ER membrane in transgenic Brassica napus plants overexpressing the oil body protein. However, these authors were unable to immunoprecipitate oleosin protein from microsomes of their transgenic plants. The difference in results may be due to the increased sensitivity afforded this study by using radiolabelled in vitro synthesised proteins, or that Sarmiento et al. [24] used a non-denaturing (native) immunoprecipitation protocol. The authors speculated that their inability to immunoprecipitate any microsomal oleosin was due to conformational changes in the protein.
2.3. Oleosin transcript associated with the rER is displaceable by Mg2+-chelation It is possible to release the rough microsomes from the attached ribosomes by stripping them with a solution containing a high concentration of EDTA, which chelates the Mg2+ required for ribosome attachment [17, 33]. This was used as an alternative method to confirm that the oleosin transcripts were associated with microsomally-bound ribosomes and that these polysomes are specifically bound to the ER-derived membranes. These rER polysomes were released from the membranes by washing with a buffer containing 25 mM EDTA, the freed ribosomes retained (after Plant Physiol. Biochem.
Figure 2. The oleosin protein is encoded by rER-associated transcripts. A, Displacement of rER-associated transcripts by chelation was carried out by incubation in the presence or absence of 25 mM EDTA. After incubation, the membranes were recovered by centrifugation, translated in a wheat germ extract and analysed as described above. Lane 1, Control rough microsomes incubated without EDTA; lane 2, rough microsomes incubated 1 h in the presence of 25 mM EDTA. B, Chelation-displaced polysomal RNA was recovered and in vitro translated in wheat germ extract. Translation products were then immunoprecipitated with anti-oleosin antisera and analysed as described above. Lane 1, Total polypeptides synthesised from the EDTA-released polysomes; lane 2; immunoprecipitation supernatant (non-precipitating proteins); lane 3, polypeptides immunoprecipitated by the oleosin antisera.
centrifugal removal of the resulting smooth ER) and their attendant mRNA transcripts extracted. This population of released RNAs was then used to programme a wheat germ cell-free in vitro translation reaction. When the translation products were analysed by SDSPAGE, it was clear that EDTA had displaced endogenous transcripts from the microsomes (figure 2 A, lane 2), compared with the in vitro translation of non-treated microsomes (figure 2 A, lane 1). Moreover, the polypeptide profile generated from in vitro translation of the EDTA-released polysomal mRNAs (figure 2 B, lane 1) was very similar to that obtained with the addition of the rough microsomes (figure 2 A, lane 1), indicating that these transcripts are indeed associated with the membrane-bound ribosomes and are displaceable by alteration of the divalent cation concentration. The polypeptides synthesised in vitro by the EDTAreleased polysomal RNA (products shown in figure 2 B, lane 1) were immunoprecipitated with the oleosin antisera, identifying a polypeptide z20 kDa (figure 2 B, lane 3), similar to those observed with the immunoprecipitation of translation products derived from the rough microsomes. Thus, the oleosin translation
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products are derived from membrane-bound ribosomes and these oleosin transcripts associate with bound ribosomes in a [Mg2+]-dependent manner, as expected for a bona fide rER-synthesised product. We consider that the results presented in figures 1 and 2 demonstrate for the first time that the oleosin protein is synthesised on bound ribosomes and cotranslationally inserted into the ER membrane.
2.4. Insertion of the oleosin protein into microsomal membranes We wished also to compare the insertion of the ‘run-on’ translated oleosin with that observed for the protease-resistant insertion of oleosin into canine pancreatic microsomal membranes [1, 16]. Translation of sunflower rough microsomes in wheat germ extract was carried out, followed by isolation of the microsomes by centrifugation through a sucrose cushion. The membranes were then subjected to protease digestion, followed by carbonate extraction and immunoprecipitation with the oleosin antibody. Unfortunately, it was not possible to immunoprecipitate a proteaseprotected oleosin fragment using this approach. An explanation for this lack of immunoprecipitation may be due to the low antigenicity of the central hydrophobic domain, resulting in a polyclonal serum that does not recognise this region. In order to verify this hypothesis, we used a set of primers to generate a PCR product (termed pCD) corresponding to the central hydrophobic domain (74 residues) of the sunflower oleosin [28]. These primers were also used separately to produce two oleosin deletions lacking either the entire C-terminal domain (pC115) or the N-terminal domain (pN40) of the protein. These three PCR products were cloned into an in vitro expression vector and expressed in a cell-free transcription/translation system, with the fulllength oleosin as a control, then immunoprecipitated using the oleosin antiserum (figure 3). The results show that although both the N-terminal (C115) and the C-terminal (N40) domains are efficiently immunoprecipitated by the antiserum (figure 3 C), the central hydrophobic domain of the oleosin is clearly not antigenic, since deletion CD was not immunoprecipitated (figure 3 A, B). Two other antisera raised against synthetic peptides based on the sequence of the central domain also failed to immunoprecipitate deletion CD (unpubl. obs.). We therefore concluded that it would not be possible to immunoprecipitate an oleosin protease-resistant fragment from the ‘run-on’ translated sunflower rER.
Figure 3. The central hydrophobic domain of the sunflower oleosin is not recognised by oleosin antisera. A, In vitro transcription/translation of full-length oleosin cDNA or template corresponding to the central hydrophobic domain. Translation products were analysed by SDS-PAGE and autoradiography. WTSO, Fulllength sunflower oleosin; CD, central domain only of sunflower oleosin. B, Immunoprecipitation of full-length and truncated oleosin translation products (as in A) was carried out. Only the WTSO (full-length protein) was immunoprecipitated, unlike the CD (central domain only) truncation. C, In vitro transcription/translation of either the full-length oleosin (WTSO), a N-terminal deletion (N40) or a C-terminal deletion (C115), followed by immunoprecipitation using the oleosin antisera indicated that both truncated forms, N40 and C115 (lacking respectively the entire N- and C-terminal domains), are efficiently precipitated. Some minor additional bands were obtained (*). These bands are likely to correspond to internal methionine codons being used as alternative start codons in the cell-free translation system.
2.5. In vitro synthesised oleosin inserts into sunflower smooth ER Another approach was therefore necessary to analyse the insertion of the oleosin into the sunflower microsomes. A coupled transcription/translation wheat germ cell-free extract was programmed with the fulllength sunflower oleosin cDNA, in the presence of EDTA-stripped-sunflower microsomal membranes. These ‘smooth’ microsomes had no associated ribosomal mRNAs (see figure 1, lane 2) and are therefore dependent on exogenous RNA (i.e. the in vitro transcribed sunflower oleosin transcript) templates. The translation products of the reaction were dual-labelled using L-[35S]-methionine and L-[3H]-leucine, and the activity of the sunflower microsomes was compared with that of canine pancreatic microsomes. After translation, the microsomes were isolated and incubated in the presence of proteinase K as previously described. This treatment generated a ∼8-kDa protected polypeptide, but only in the presence of sunflower or dog microsomes (figure 4 A, lanes 4, 6) vol. 37 (6) 1999
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Figure 4. N-terminal radio-sequencing of a membrane-insertedprotease resistant oleosin fragment. A, In vitro L-[35S]-methionineand L-[3H]-leucine-labelled transcription/translation of the full-length oleosin construct in the absence of (lanes 1, 2) or in the presence of (lanes 3, 4) ‘smooth’ sunflower microsomes, or canine microsomes (lanes 5, 6) was carried out, membranes purified by flotation, proteinase K-digested and carbonate-extracted. The resulting (protease-protected) polypeptides were identified by SDS-PAGE and fluorography. An 8-kDa protected fragment was obtained with both the sunflower and the canine microsomes (lanes 4, 6). The peptide marked with an arrow-head was transferred on a nitrocellulose membrane and analysed by N-terminal radio-sequencing. B, N-terminal radio-sequence derived from the Edman degradation (sixteen cycles) of the protease-resistant oleosin fragment. Each fraction was scintillation-counted for both [35S] and [3H] labels. No signal was obtained in the emission range for [35S] but four peaks were detected in the [3H] emission range. The corresponding position of these tritiated leucines (underlined) in the amino acid sequence of the sunflower oleosin [28] is also indicated.
whereas no protease-resistant polypeptide was observed in the absence of membranes (figure 4 A, lane 2). It is of interest to note that the protease-protected oleosin polypeptides generated by either sunflower or canine microsomes were of similar mobility, and that the predicted size of this polypeptide is very close to that observed for protease-treated oil bodies [14, 30].
2.6. Identification of the protease-protected oleosin domain by radio-sequencing Having confirmed that the sunflower oleosin was inserted into sunflower microsomal membranes in a carbonate- and protease-resistant manner, we wished Plant Physiol. Biochem.
to define the precise position of the protease-protected domain. Previously, Abell et al. [1] used a series of Nand C-terminal deletions to map the position of the protease-resistant domain of an Arabidopsis oleosin. Since this method is dependent on the resolution and sizing of small polypeptide fragments on polyacrylamide gels, we utilised a more precise approach. The (dual-labelled) in vitro synthesised protease-protected polypeptides were separated by polyacrylamide gel electrophoresis, blotted onto a nitrocellulose membrane and identified by autoradiography. The appropriate region of the membrane was then excised and subjected to automated Edman degradation radiosequencing [2]; sixteen cycles were performed and each cycle fraction was scintillation-counted for the presence of both [35S]- and [3H]-label. This showed that there was no detectable [35S]-methionine label within the sixteen cycles counted (corresponding to the first sixteen residues of the protease-protected portion of the oleosin). However, a number of signals were obtained from the [3H]-label as shown in figure 4 B. Four distinct peaks were identified, corresponding to the incorporation of [3H]-leucine into the oleosin protected fragment at positions 1, 7, 10 and 15. The amino acid sequence of the sunflower oleosin contains only one sequence which matches this pattern [LeuX5-Leu-X2-Leu-X4-Leu] which is just inside the start of the central hydrophobic domain. This region also lacks methionine residues, confirming the lack of recovered [35S]-methionine. Thus, the N-terminus of the protease-protected fragment of the sunflower oleosin can be mapped as starting at Leu50, as shown in figure 4 B. Although the protected fragment was derived from sunflower microsomal membranes, an identical pattern of incorporation was also observed with the protease-protected fragment from canine membranes (data not shown). This indicates that oleosin is likely to insert into the different microsomal membranes in a similar manner, and also validates the previous use of canine pancreatic microsomes for studies on the targeting of plant oleosins.
2.7. Comparison of oil body and microsomal membrane protease-protected oleosin fragments We have also recently determined the N-terminal sequence of the protected portion of the oleosin after protease-digestion of safflower oil bodies and found this to be Leu-Ala-Gly-Gly-Ser [14]. This motif is highly conserved among a number of dicot oleosins, such as those from Arabidopsis, rape, almond and citrus, and is found just inside the start of the central hydrophobic domain of the protein. Thus, it was
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Figure 5. Position of the N-terminal sequence of the protease-protected fragment within the sunflower oleosin. A comparison of the deduced amino acid sequences of the central domains of oleosins from sunflower, Arabidopsis, soybean, cotton, carrot and Brassica napus. The position of the start of the protease-protected fragment derived from microsomal insertion is indicated with a solid arrowhead and the position of the tritiated leucines identified by radio-sequencing indicated by dots. The start of the protease-protected fragment derived from oil bodies [14] is indicated with the open arrow, and the start of the hydrophobic domains indicated by the line.
possible to compare the N-termini of proteaseprotected fragments from oleosins inserted into microsomes or oil bodies. This showed (figure 5) that the protected fragments differ slightly in the position of their N-termini, with the sequence of the microsomal fragments starting one residue before that deduced for the oil body fragments. In addition, in both cases, the protease-resistant fragments do not start at the junction of the N-terminal domain and the central hydrophobic domain that has been predicted by modelling [11] or deletion analysis [1], but actually inside this central region. Importantly, it is also clear that the N-termini of these protease-protected fragments of the oleosin represent the start of the highly conserved central domain, as opposed to the start of the hydrophobic region per se. In the case of the sunflower oleosin, the hydrophobic region starts at Ile43 which is seven residues before the N-terminus of the protected fragment at Leu50. These data will therefore be useful in modelling the topology of the oleosin in either microsomal membranes or oil bodies.
3. CONCLUSION We provide evidence that the oleosin transcript is associated with the rough ER and that the oleosin protein is synthesised on bound ribosomes and cotranslationally inserted into the microsomal membrane. We have also determined the N-terminal sequence of a protease-protected fragment of microsomally inserted-oleosin, showing that not all of the central hydrophobic domain of the protein is inserted into the membrane. A similar sequence was observed for the oleosin in oil bodies.
4. METHODS 4.1. Plant material Sunflower plants (Helianthus annuus L., cv. Sunbred 246) were grown under greenhouse conditions with a photoperiod of 16 h light at 25 °C and 8 h dark at 18 °C. Seeds were harvested 15–17 d after flowering, corresponding to the mid-maturation stage. Chemicals were purchased from Sigma Chemicals (Poole, UK) unless otherwise stated. Radiochemicals were obtained from Amersham International (Amersham, UK). 4.2. Preparation of ER-derived microsomal membranes Embryonic tissue (1–2 g) was extracted from the developing seeds and homogenised on ice in 1–2 mL extraction buffer containing 0.6 M RNAse-free sucrose in 50 mM Hepes/KOH (pH 7.5), 10 mM KCl, 62.5 mM potassium acetate, 5 mM EGTA, 5 mM DTT and 2 units⋅mL–1 RNAsin (Promega) using a sterile mortar and pestle. Two variants of this buffer were used at this stage, buffer A supplemented with 5 mM MgCl2 and 1 % BSA (w/v) for extraction of rough microsomes or buffer B supplemented with 1 mM MgCl2, 5 mM EDTA and 1 % BSA (w/v) for extraction of smooth microsomes. The volume was increased to 12 mL with the appropriate extraction buffer and the cell homogenate was centrifuged at 1 000 × g for 15 min at 4 °C using a FO650 rotor (Beckman) to pellet the cell debris. The fat pad formed by the oil bodies was removed, the supernatant further centrifuged at 100 000 × g for 1 h at 4 °C in a TST 41.14 swing-out rotor (Kontron Instruments). The pellet was resuspended with 1 mL resuspension buffer C (identical to buffer A but containing 0.25 M sucrose), per g of tissue, and kept on ice until required. vol. 37 (6) 1999
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4.3. EDTA stripping of microsomes Microsomal membranes were extracted as described above with a buffer identical to buffer A but containing 100 µg⋅mL–1 PMSF instead of BSA. After the final 100 000 × g centrifugation, the microsomal pellet was resuspended in the same extraction buffer and the volume increased to 12 mL with a polyribosomereleasing buffer identical to buffer B but containing 100 µg⋅mL–1 PMSF instead of BSA and supplemented with 25 mM EDTA. This suspension was incubated 1 h at 4 °C with gentle mixing. The membranes were then pelleted again at 10 000 × g for 1 h at 4 °C as previously described. This washed membrane pellet was resuspended in 1 mL normal resuspension buffer C (BSA-containing) per g of tissue and kept on ice until required. The 100 000 × g supernatant from the washed microsomes was extracted with an equal volume of unbuffered phenol-chloroform and twice extracted with an equal volume of chloroform. The RNA was precipitated from the final supernatant with 2 M lithium acetate at –80 °C. After thawing on ice, the solution was centrifuged at 10 000 × g at 4 °C for 30 min using a JA-25 rotor (Beckman) and the pellet resuspended in 1 mL sterile water. The RNA was again precipitated with ethanol, desalted with 70 % ethanol and dissolved in water at a concentration of 1–2 mg⋅mL–1. This RNA stock was flash-frozen in liquid nitrogen and stored at –80 °C.
4.4. Oleosin truncated constructs A N-terminal deletion of an oleosin cDNA (pSO5; [28]) was generated by PCR using primer N40 (5’-GCCCATGGGCAAGATAATGGTCATCATGGCC3’) introducing a NcoI site (underlined), and the T7 terminator primer. A C-terminal deletion was generated similarly using primer C115 (5’-GCCTA ATACGACAACGAGCTTAACCC-3’) introducing a stop codon (underlined), and the T7 promoter primer. To allow a directional cloning, both fragments were digested with NcoI and ligated in a NcoI cut pCite T-vector system (Novagen) using a T4 DNA ligase (Promega, UK). The N-terminal truncation pN40 lacks the entire N-terminal amphipathic domain of the protein. The C-terminal truncation pC115 lacks the entire C-terminal hydrophilic domain. A construct lacking both the N- and C-terminal domains was produced using both primer N40 and C115 together and cloned as described. This N- and C-terminal truncation pCD corresponds to the central hydrophobic domain of the protein. Plant Physiol. Biochem.
4.5. In vivo translation using a wheat germ extract For translation of the microsome associated mRNAs, 50 µL reactions were carried out and the product was stored for up to 1 week at –20 °C. For each reaction, 5 µL membrane suspension or purified RNA was used as a template. In the case of released RNA, the stock solution was initially denatured by heating at 65 °C for 10 min. Then, 32.5 µL premix was added to the template and the reaction volume made up to 50 µL with sterile water. The premix was such that the final concentration of the reagents was 50 % wheat germ extract (Promega), 80 µM amino acid mixture (minus methionine), 0.8 U⋅µL–1 RNAsin and 1.85⋅107 Bq⋅mL–1 L-[35S]-methionine. These reactions were incubated for 1 h at 25 °C. In some cases, the translation was carried out using a centrifuged wheat germ extract in which microsomal membranes and free ribosomes were removed. For this purpose, 200 µL aliquots of wheat germ were centrifuged at 290 000 × g for 1 h at 4 °C in 8 × 34 mm polycarbonate tubes (Beckman) using a Beckman TLA100 rotor. Full-length and truncated clones of the oleosin cDNA were expressed in a cell-free coupled transcription/translation system (Promega) using the T7 RNA polymerase. Reactions were carried out in 10 µL following the manufacturer’s instructions and labelled in presence of 1.85⋅107 Bq⋅mL–1 L-[35S]methionine. After translation, the samples were either analysed further or mixed with an equal volume of 2× SDS-PAGE sample buffer. For the protease-protection assay, the full-length construct pSO5 was expressed in a coupled transcription/translation wheat germ extract but 20 µL reactions were translated in the presence or absence of either 2 µL canine microsomes or EDTAwashed sunflower microsomes. The products were labelled with 1⋅107 Bq⋅mL–1 L-[35S]-methionine and 1.85⋅107 Bq⋅mL–1 L-[3H]-leucine. After translation, the reaction volume was split in two microfuge tubes and one retained as non-digested control. In the other tube, proteinase K was added to give a final concentration of 200 µg⋅mL–1 (water instead of proteinase K was added in the control). The samples were then incubated for 30 min on ice and the reaction terminated by addition of PMSF to a final concentration of 2 mM and incubated for a further 5 min on ice. 4.6. Isolation and purification of the microsomes from the translation mixture After in vitro translation or proteinase K digestion, the samples were diluted with an equal volume of 0.5 M potassium acetate and the microsomes purified
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through a 0.6-M sucrose cushion containing 0.5 M potassium acetate, 5 mM MgCl2 and 50 mM HepesKOH (pH 7.9). The samples were centrifuged at 290 000 × g for 10 min at 4 °C using polycarbonate centrifuge tubes and a TLA-100 rotor as described previously. The microsomal pellets were resuspended in 50 µL buffer containing 0.25 M sucrose, 0.1 M potassium acetate, 5 mM MgCl2, 50 mM Hepes-KOH (pH 7.9) and sodium carbonate was added to a final concentration of 0.1 M (pH 11) [5]. The samples were incubated for 30 min on ice and the microsomes purified as described above.
4.7. Immunoprecipitation After translation or isolation and purification of the microsomes, the volume of each sample was increased to 500 µL with RIPA buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.1 % SDS (w/v), 1 % Nonidet P-40 (v/v), 0.5 % deoxycholic acid (w/v)) [13]. Oleosin polyclonal antiserum (3 µL) was added to each sample and incubated at 4 °C with gentle agitation for 1 h. Pre-swollen protein A-Sepharose (15 mg, Sigma) was added in each sample and the sample incubated overnight at 4 °C with gentle mixing. After binding, the beads were pelleted in a microcentrifuge, the supernatant removed, and the antigen/antibody/ protein-A complex gently washed by inversion with 500 µL RIPA buffer for 20 min at 4 °C. Several rounds of pelleting and washing in RIPA buffer were carried out, followed by a final wash with 10 mM Tris/HCl (pH 7.5), 0.1 % Nonidet P-40 (v/v). After centrifugation, the pellet was resuspended in 30 µL 2× SDSPAGE sample buffer and the mixture heated at 100 °C for 3 min to ensure the release of the protein from the protein A-Sepharose/IgG complex. When the supernatant from the initial incubation was retained for analysis, avoiding contaminations by the beads, the protein was precipitated with 10 % TCA for 30 min on ice. After centrifugation for 10 min at 13 000 × g, the pellets were washed twice with 1 mL 80 % acetone to remove the TCA and resuspended in 30 µL SDSPAGE sample buffer.
4.8. Proteinase protection assay After translation, in presence or absence of canine microsomes or EDTA-washed sunflower microsomes, the reaction volume was split in two microfuge tubes and one kept as non-digested control. In the other tube, proteinase K was added to give a final concentration of 200 µg⋅mL–1 (water instead of proteinase K was added in the control). The samples were then incubated on
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ice for 30 min and the reaction terminated by addition of PMSF to a final concentration of 2 mM and incubated for a further 5 min on ice.
4.9. Analysis by SDS-PAGE The proteins after either in vitro translation or immunoprecipitation were resolved by SDS-PAGE using a tricine buffer system based on that of Schagger and von Jagow [25]. The electrophoresis system consisted of a 15 and 4.5 % polyacrylamide in the separating gel and the stacking gel, respectively, and was run at 100 V until the loading dye ran off. After electrophoresis, the gels were fixed with water/ methanol/acetic acid (80/10/10 v/v/v), soaked in AmplifyTM (Amersham), dried and the products were detected by fluororadiography using BiomaxTM film (Kodak).
4.10. Radio-sequencing This was carried out essentially as described by Anderson and Gray [2]. N-terminal sequencing was carried out in the Department of Biochemistry, University of Bristol.
Acknowledgments IACR-Long Ashton Research Station receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. DJL was the recipient of a BBSRC-ROPA award. FB was funded by the French Ministry of Higher Education and Research. We thank Dr W. Mawby (Department of Biochemistry, University of Bristol) for carrying out the radio-sequencing. Canine pancreatic microsomes were the kind gift of Dr Steve High, University of Manchester.
REFERENCES [1] Abell B.M., Holbrook L.A., Abenes M., Murphy D.J., Hills M.J., Moloney M.M., Role of the proline knot motif in oleosin endoplasmic reticulum topology and oil body targeting, Plant Cell 9 (1997) 1481–1493. [2] Anderson C.M., Gray J., Cleavage of the precursor of pea chloroplast cytochrome-f by leader peptidase from Escherichia coli, FEBS Lett. 280 (1991) 383–386. [3] Ceriotti A., Pedrazzini E., Desilvestris M., Vitale A., Import into the endoplasmic reticulum, Methods Cell Biol. 50 (1995) 295–308. vol. 37 (6) 1999
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[4] Cummins I., Hills M.J., Ross J.H.E., Hobbs D.H., Watson M.D., Murphy D.J., Differential, temporal and spatial expression of genes involved in storage oil and oleosin accumulation in developing rapeseed embryos: implications for the role of oleosins and the mechanisms of oil-body formation, Plant Mol. Biol. 23 (1993) 1015–1027. [5] Fujiki Y., Hubbard A.L., Fowler S., Lararow P.B., Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplamic reticulum, J. Cell Biol. 93 (1982) 97–102. [6] Galili G., Sengupta-Gopalan C., Ceriotti A., The endoplasmic reticulum of plant cells and its role in protein maturation and biogenesis of oil bodies, Plant Mol. Biol. 38 (1998) 1–29. [7] Herman E.M., Immunogold-localization and synthesis of an oil-body membrane protein in developing soybean seeds, Planta 172 (1987) 336–345. [8] Herman E.M., Cell and molecular biology of seed oil development, in: Kigel J., Galili G. (Eds.), Seed development and germination, Marcel Dekker Inc., New York, 1995, pp. 195–214. [9] Hills M.J., Watson M.D., Murphy D.J., Targeting of oleosins to the oil bodies of oilseed rape (Brassica napus L.), Planta 189 (1993) 24–29. [10] Huang A.H.C., Oil bodies and oleosins in seeds, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43 (1992) 177–200. [11] Huang A.H.C., Oleosins and oil bodies in seeds and other organs, Plant Physiol. 110 (1996) 1055–1061. [12] Lacey D.J., Hills M.J., Heterogeneity of the endoplasmic reticulum with respect to lipid synthesis in developing seeds of Brassica napus L., Planta 199 (1996) 545–551. [13] Lacey D.J., Beaudoin F., Dempsey C.E., Shewry P.R., Napier J.A., The accumulation of triacylglycerols within the endoplasmic reticulum of developing seeds of Helianthus annus, Plant J. 17 (1999) 397–405. [14] Lacey D.J., Wellner N., Beaudoin F., Napier J.A., Shewry P.R., Secondary structure of oleosins in oil bodies isolated from seeds of safflower (Carthamus tinctorius L.) and sunflower (Helianthus annuus L.), Biochem. J. 334 (1998) 469–477. [15] Li X., Franceschi V.R., Okita T.W., Segregation of storage protein mRNAs on the rough endoplasmic reticulum membranes of rice endosperm cells, Cell 72 (1993) 869–879. [16] Loer D.S., Herman E.M., Cotranslational integration of soybean (Glycine max) oil body membrane protein oleosin into microsomal membranes, Plant Physiol. 101 (1993) 993–998. [17] Lord M.J., Isolation of endoplasmic reticulum: general principles, enzymatic markers and endoplasmic reticulum bound polysomes, Methods Enzymol. 148 (1987) 576–590. [18] Lord M.J., Kagawa T., Moore T.S., Beevers H., Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm, J. Cell Biol. 57 (1973) 659–667.
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[19] Mazhar H., Quayle R., Fido R.J., Stobart A.K., Napier J.A., Shewry P.R., Synthesis of storage reserves in developing seeds of sunflower, Phytochemistry 48 (1998) 429–432. [20] Millichip M., Tatham A.S., Jackson F., Griffiths G., Shewry P.R., Stobart A.K., Purification and characterization of oil-bodies (oleosomes) and oil-body boundary proteins (oleosins) from the developing cotyledons of sunflower (Helianthus annuus L.), Biochem. J. 314 (1996) 333–337. [21] Murphy D.J., Structure, function and biogenesis of storage lipid bodies and oleosins in plants, Prog. Lipid Res. 32 (1993) 247–280. [22] Napier J.A., Stobart A.K., Shewry P.R., The structure and biogenesis of plant oil bodies: the role of the ER membrane and the oleosin class of proteins, Plant Mol. Biol. 31 (1996) 945–956. [23] Qu R., Wang S.M., Lin Y.H., Vance V.B., Huang A.H.C., Characteristics and biosynthesis of membrane proteins of lipid bodies in the scutella of maize (Zea mays L.), Biochem. J. 234 (1986) 57–65. [24] Sarmiento C., Ross J.H.E., Herman E., Murphy D.J., Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds, Plant J. 11 (1997) 783–796. [25] Schagger H., von Jagow G., Tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis for the separation of proteins in the range 1-100 kD, Anal. Biochem. 166 (1987) 368–379. [26] Shewry P.R., Napier J.A., Tatham A.S., Seed storage proteins: structure and biosynthesis, Plant Cell 7 (1995) 945–956. [27] Stobart A.K., Stymne S., Hoglund S., Safflower microsomes catalyse oil accumulation in vitro: a model system, Planta 169 (1986) 33–37. [28] Thoyts P.J.E., Millichip M.I., Stobart A.K., Griffiths W.T., Shewry P.R., Napier J.A., Expression and in vitro targeting of a sunflower oleosin, Plant Mol. Biol. 29 (1995) 403–410. [29] Thoyts P.J.E., Napier J.A., Millichip M., Stobart K.A., Griffiths T.W., Tatham A.S., Shewry P.R., Characterisation of a sunflower seed albumin which associates with oil bodies, Plant Sci. 118 (1996) 119–125. [30] Tzen T.C., Huang A.H.C., Surface structure and properties of plant seed oil bodies, J. Cell Biol. 117 (1992) 327–335. [31] Tzen J.T.C., Cao Y., Laurent P., Ratnayake C., Haung A.H.C., Lipids, proteins, and structure of seed oil bodies from diverse species, Plant Physiol. 101 (1993) 267–276. [32] Tzen J.T.C., Lai Y., Chan K., Huang A.H.C., Oleosin isoforms of high and low molecular weights are present in the oil bodies of diverse seed species, Plant Physiol. 94 (1990) 1282–1289. [33] Walter P., Blobel G., Preparation of microsomal membranes for cotranslational protein translocation, Methods Enzymol. 96 (1982) 84–96.
The biogenesis of the plant seed oil body: Oleosin protein is synthesised by ER-bound ribosomes Frédéric Beaudoin, Dominic J. Lacey, Johnathan A. Napier* IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton Bristol BS41 9AF, UK * Author to whom correspondence should be addressed (fax +44 1275 394281; e-mail [email protected])
(Received December 15, 1998; accepted March 29, 1999) Abstract — An in vitro system was developed to demonstrate that the oleosin protein is synthesised on bound ribosomes and co-translationally inserted into the endoplasmic reticulum (ER) membrane. Microsomes were isolated with their associated bound ribosomes and transcripts from the developing embryos of sunflower (Helianthus annuus L.), and used to programme a cell-free translation system. The presence of the oleosin within the population of polypeptides synthesised by these rough membranes was demonstrated by immunoprecipitation with a specific oleosin antibody. Displacement of the bound ribosomes by the chelation of Mg2+ was also shown to displace the oleosin transcript. In vitro synthesised oleosin was shown to insert into sunflower microsomal membranes, with a portion of the protein being resistant to protease digestion. Radio-sequencing was used to determine the N-terminus of this fragment, providing new insights into the topology of the oleosin protein. © Elsevier, Paris Endoplasmic reticulum membrane / oleosin / oilseeds / seed development ER, endoplasmic reticulum / SRP, signal recognition particle / TAG, triacylglycerol
1. INTRODUCTION Oleosins are a unique class of proteins found exclusively in oil bodies of lipid-storing tissues such as seeds and pollen [10, 21, 22]. Oleosins are usually in the size range of 15–26 kDa, and have a characteristic three-domain structure. This takes the form of a highly conserved hydrophobic central domain of usually seventy residues, flanked by two amphipathic regions of a more variable nature. In most developing oilseeds, the oleosin protein is localised to the surface of the oil body (the site of triacylglycerol [TAG] deposition), and is postulated to play a role in both the biogenesis of the organelle [7, 8] and also in the prevention of aggregation of the storage lipid during seed maturation and desiccation [4]. Although the oil body is a relatively simple organelle, being made up of a TAG interior and an oleosin-phospholipid shell, the mechanisms by which it is formed are unclear and has been the subject of a degree of controversy [10, 21, 22]. What is clear is that storage lipid in the form of TAG is synthesised by the ER membrane [13, 27], probably between the leaflets of the membrane bilayer. This has led to speculation Plant Physiol. Biochem., 0981-9428/99/6/© Elsevier, Paris
that the oil body might arise as a result of concomitant synthesis of TAG and oleosin by the ER, with the oleosin being targeted and co-translationally inserted into the membrane by the signal recognition particle (SRP) pathway [22]. Although there is still some dispute as to the temporal regulation of TAG synthesis versus oleosin synthesis [4, 31], this SRP-dependent hypothesis has become widely accepted [1, 6]. This has been enhanced by studies on the targeting and deposition of the oleosin protein as a means of investigating oil body biogenesis. Initial studies by two independent groups indicated that in vitro synthesised oleosin was unable to insert into isolated oil bodies, but was able to insert into microsomal membrane extracts [9, 16]. Moreover, the in vitro synthesis of the oleosin was stimulated by the addition of microsomes, indicative of co-translational insertion. More recently, we have shown that this effect is due to the interaction between the oleosin and SRP [28] causing translational arrest which is relieved by the presence of the microsomal SRP receptor. However, all these in vitro studies made use of canine pancreatic microsomal membranes and in vitro transcribed (from cloned cDNA templates) mRNAs which, although a
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model system for the study of ER membrane protein targeting, is clearly non-homologous for developing oilseeds. Ultrastructural studies have been carried out to try and identify any association between the ER and the oil body and, whilst a number of authors have reported this phenomenon (see [8] for a review), others have failed to observe any such connections. Therefore, if the biogenesis of oil bodies is to be studied in detail, it is essential to develop novel methods. Stobart et al. [27] reported in vitro accumulation of TAG to high levels, using a microsomal membrane preparation derived from safflower cotyledons. The possibility of combining in vitro TAG synthesis with that of oleosin, resulting in in vitro synthesised oil bodies, might provide insights into organellar biogenesis [13, 22]. To facilitate the assembly of such a system, we decided to investigate the association of oleosin transcripts in vivo with membrane-bound ribosomes. Previously, Qu et al. [23] reported that the oleosin protein (or protein L3 as it was then designated) was synthesised by membrane-bound polysomes. This was based on their observation that cell-free in vitro translation products of RNA extracted from bound polysomes contained a polypeptide of similar mobility to that of the oleosin protein. This initial report has subsequently been cited as demonstrating the synthesis of oleosin on rough ER membranes (see, for example [1, 8, 9]) However, careful reading of the original data presented by Qu et al. [23] indicates that the identity of the putative rER-derived oleosin (L3) translation product was not confirmed immunologically, with identification based solely on electrophoretic mobility. Moreover, the abundance of this putative oleosin transcript among total ER-bound polyribosome mRNAs casts some uncertainty on its identity, since the data indicated that the translation product identified by Qu et al. [23] was the major, if not the only, protein synthesised by bound ribosomes. In contrast, other studies of a number of related species showed that among total protein expressed in developing seeds, the seed storage proteins are by far the most abundant class of polypeptide (see [26] for a review). It is also noteworthy that other workers have shown that oleosins are only minor components (< 5 %) of proteins immunoprecipitated from in vitro translated mRNA isolated from developing seeds [9]. These seed storage proteins are synthesised exclusively by ER-bound polyribosomes, prior to their entry into the plant secretory system [26]. These observations clearly indicate that further studies were required to verify the synthesis of oleosin by bound ribosomes on the rER. Therefore, we isoPlant Physiol. Biochem.
lated microsomal membranes from developing sunflower cotyledons with or without their associated ribosomes, based on the Mg2+ requirement for ribosome binding. These microsomes were then used to programme a wheat germ cell-free in vitro translation system, and resulting polypeptides were characterised by immunoprecipitation using a polyclonal antiserum raised against sunflower oil-bodies. This demonstrated that the sunflower oleosin transcripts are among the population of rER-associated mRNAs and that this protein is translated on bound ribosomes. Moreover, we also show that oleosin protein synthesised in vitro inserts into sunflower microsomal membranes in a carbonate-extraction-resistant manner, and proteaseprotection studies revealed polypeptides resistant to digestion of a similar size to those obtained with protease-digested sunflower-oil bodies.
2. RESULTS AND DISCUSSION 2.1. The isolation of transcripts associated with the rER of sunflower microsomes Previous studies of the insertion of oleosins into microsomal membranes have used canine pancreatic microsomes as a substrate [9, 16, 28]. We wished to carry out in vitro oleosin membrane insertion assays in a more homologous system, using not only plantderived microsomal membranes, but also the endogenous oleosin mRNAs associated with the polyribosomes bound to the membranes. Therefore, we isolated ER-derived microsomes from developing cotyledons of sunflower. Plant ER-derived microsomes can be isolated by sucrose density gradient centrifugation and separated into smooth- and rough-surfaced microsomes in presence of Mg2+ because of their different equilibrium densities [3, 17, 18, 33], the latter having a greater density as a result of the associated ribosomes. However, it has been demonstrated more recently that, in developing embryos of Brassica napus, the ER is heterogeneous with respect to its ability to synthesise storage lipid [12, 13] with the enzymes of TAG synthesis being associated with lighter membrane fractions with equilibrium densities ranging from 12 to 28 % sucrose (w/w), presumably due to their increased TAG content. Microsomes extracted from mid-maturation embryos of sunflower, in the presence or in the absence of EDTA, generated smooth and rough microsomes, respectively, and were added to a wheat germ cell-free extract to allow the in vitro translation of any endogenous mRNAs associated with the microsomal prepa-
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Figure 1. SDS-PAGE analysis of in vitro translated microsomes preparations and the polypeptide complement of associated rER-bound transcripts. A, Membranes were translated in a wheat germ system, the products separated by SDS-PAGE and radiolabelled proteins detected by autoradiography. Lane 1, Microsomes extracted in presence of 5 mM MgCl2 without EDTA; lane 2, microsomes extracted in presence of 1 mM MgCl2 and 5 mM EDTA; lane 3, wheat germ negative control incubated without membrane. B, Sunflower rough microsomes extracted in the presence of 5 mM MgCl2 (lanes 1, 3, 5) or in vitro transcribed preprolactin mRNAs (lanes 2, 4, 6) were translated using either: (a) normal wheat germ extract as positive control; (b) the 290 000 × g supernatant of wheat germ extract centrifuged for 1 h; (c) water added to the translation mixture instead of wheat germ as negative control. The products of the reactions were analysed as described previously. C, After in vitro translation, the rER membranes were resuspended and immunoprecipitated with either anti-oleosin antisera (lane 1) or pre-immune sera (lane 2) and analysed by SDS-PAGE and autoradiography. The positions of molecular mass markers is indicated.
rations. The radiolabelled translation products were analysed by SDS-PAGE and fluorography (figure 1 A). A negative control (no membrane added) was included to identify any endogenous wheat germ mRNA. After in vitro translation, the rough microsomes synthesised a number of proteins ranging from 15 to 90 kDa (figure 1 A, lane 1), whereas almost no labelled protein was detectable in the sample translated in the presence of smooth microsomes (figure 1 A, lane 2). This confirmed that in the presence of 5 mM EDTA, polysomes were released from the sunflower microsomes, and that the free cytosolic ribosomes were not pelleted with the membranes. The wheat germ control (figure 1 A, lane 3) gave a very faint band of 32 kDa, often observed with this in vitro translation system in the absence of exogenous template mRNAs. The bands obtained with the rough microsome preparation are the products of transcripts associated with the ribosomal population bound to these membranes (i.e. ribosome complexes which have been associated in vivo with the rER by virtue of a SRP-signal sequence present in the polypeptide nascent chain). The darkest bands correspond to the most abundant mRNAs associated with the microsomal-bound ribosomes, and probably represent seed storage proteins, such as 11S globulin subunits (helianthenins) and 2S albumins, which are highly transcribed in sunflower during mid-stage seed development [19, 29]. Storage proteins are well known
to be associated with, and synthesised on, membranebound polysomes, since they possess signal sequences to direct them into the ER lumen [6, 15]. However, transcripts encoding for oleosin are also synthesised at this stage of sunflower cotyledon development [28], and a number of polypeptides of similar mobility (∼20 kDa) to sunflower oleosins [32] are present. To confirm the rER-derived nature of the polypeptide profile, a membrane- and ribosome-free wheat germ extract was prepared by high speed centrifugation. The result obtained (figure 1 B) clearly demonstrated that whilst both the rER-associated transcripts, and control preprolactin mRNA translated efficiently in a normal wheat germ extract (figure 1 B, lanes 1, 2), only the microsome-associated polyribosomes (figure 1 B, lane 3), but not preprolactin (figure 1 B, lane 4), were translated in the centrifuged (i.e. cytosolic ribosomefree) wheat germ extract. The negative controls (figure 1 B, lanes 5, 6) did not show any background. This clearly shows that the polypeptide profile generated by our in vitro translation of sunflower rough microsomes was a result of the presence of transcripts associated in vivo with the rER.
2.2. Immunoprecipitation of oleosin from in vitro translated sunflower rER In contrast to the study of Qu et al. [23], a number of proteins were present in the in vitro translation vol. 37 (6) 1999
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products programmed by membrane-bound polysomal mRNAs indicating that a precise method was required to determine if the oleosin was indeed represented in this polypeptide complement. Therefore, the in vitro translation products of reactions programmed with rough (bound polysomes) sunflower microsomes (i.e. figure 1 B, lane 3) were immunoprecipitated with either a polyclonal antiserum to the sunflower oil body proteins [20] or pre-immune serum; this immunoprecipitation step was carried out after the microsomes and their associated translation products were purified through a sucrose-step gradient and carbonateextracted. The results obtained (figure 1 C) clearly show the immunoprecipitation of a z20-kDa polypeptide, the expected size of the sunflower oleosin (lane 1) [20, 32]. This band was not observed in the case of the pre-immune sera (lane 2). Thus, the sunflower oleosin was indeed synthesised by microsomal-bound ribosomes and, co-translationally inserted into the ER membrane in a carbonate-resistant manner [5]. The observation of oleosin insertion into plant microsomal membranes complements previous results obtained from in vitro insertion of oleosin into pancreatic canine microsomes [1, 9, 16, 28]. Interestingly, a recent study by Sarmiento et al. [24] demonstrated that oleosin protein was immunolocalised to regions of the ER membrane in transgenic Brassica napus plants overexpressing the oil body protein. However, these authors were unable to immunoprecipitate oleosin protein from microsomes of their transgenic plants. The difference in results may be due to the increased sensitivity afforded this study by using radiolabelled in vitro synthesised proteins, or that Sarmiento et al. [24] used a non-denaturing (native) immunoprecipitation protocol. The authors speculated that their inability to immunoprecipitate any microsomal oleosin was due to conformational changes in the protein.
2.3. Oleosin transcript associated with the rER is displaceable by Mg2+-chelation It is possible to release the rough microsomes from the attached ribosomes by stripping them with a solution containing a high concentration of EDTA, which chelates the Mg2+ required for ribosome attachment [17, 33]. This was used as an alternative method to confirm that the oleosin transcripts were associated with microsomally-bound ribosomes and that these polysomes are specifically bound to the ER-derived membranes. These rER polysomes were released from the membranes by washing with a buffer containing 25 mM EDTA, the freed ribosomes retained (after Plant Physiol. Biochem.
Figure 2. The oleosin protein is encoded by rER-associated transcripts. A, Displacement of rER-associated transcripts by chelation was carried out by incubation in the presence or absence of 25 mM EDTA. After incubation, the membranes were recovered by centrifugation, translated in a wheat germ extract and analysed as described above. Lane 1, Control rough microsomes incubated without EDTA; lane 2, rough microsomes incubated 1 h in the presence of 25 mM EDTA. B, Chelation-displaced polysomal RNA was recovered and in vitro translated in wheat germ extract. Translation products were then immunoprecipitated with anti-oleosin antisera and analysed as described above. Lane 1, Total polypeptides synthesised from the EDTA-released polysomes; lane 2; immunoprecipitation supernatant (non-precipitating proteins); lane 3, polypeptides immunoprecipitated by the oleosin antisera.
centrifugal removal of the resulting smooth ER) and their attendant mRNA transcripts extracted. This population of released RNAs was then used to programme a wheat germ cell-free in vitro translation reaction. When the translation products were analysed by SDSPAGE, it was clear that EDTA had displaced endogenous transcripts from the microsomes (figure 2 A, lane 2), compared with the in vitro translation of non-treated microsomes (figure 2 A, lane 1). Moreover, the polypeptide profile generated from in vitro translation of the EDTA-released polysomal mRNAs (figure 2 B, lane 1) was very similar to that obtained with the addition of the rough microsomes (figure 2 A, lane 1), indicating that these transcripts are indeed associated with the membrane-bound ribosomes and are displaceable by alteration of the divalent cation concentration. The polypeptides synthesised in vitro by the EDTAreleased polysomal RNA (products shown in figure 2 B, lane 1) were immunoprecipitated with the oleosin antisera, identifying a polypeptide z20 kDa (figure 2 B, lane 3), similar to those observed with the immunoprecipitation of translation products derived from the rough microsomes. Thus, the oleosin translation
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products are derived from membrane-bound ribosomes and these oleosin transcripts associate with bound ribosomes in a [Mg2+]-dependent manner, as expected for a bona fide rER-synthesised product. We consider that the results presented in figures 1 and 2 demonstrate for the first time that the oleosin protein is synthesised on bound ribosomes and cotranslationally inserted into the ER membrane.
2.4. Insertion of the oleosin protein into microsomal membranes We wished also to compare the insertion of the ‘run-on’ translated oleosin with that observed for the protease-resistant insertion of oleosin into canine pancreatic microsomal membranes [1, 16]. Translation of sunflower rough microsomes in wheat germ extract was carried out, followed by isolation of the microsomes by centrifugation through a sucrose cushion. The membranes were then subjected to protease digestion, followed by carbonate extraction and immunoprecipitation with the oleosin antibody. Unfortunately, it was not possible to immunoprecipitate a proteaseprotected oleosin fragment using this approach. An explanation for this lack of immunoprecipitation may be due to the low antigenicity of the central hydrophobic domain, resulting in a polyclonal serum that does not recognise this region. In order to verify this hypothesis, we used a set of primers to generate a PCR product (termed pCD) corresponding to the central hydrophobic domain (74 residues) of the sunflower oleosin [28]. These primers were also used separately to produce two oleosin deletions lacking either the entire C-terminal domain (pC115) or the N-terminal domain (pN40) of the protein. These three PCR products were cloned into an in vitro expression vector and expressed in a cell-free transcription/translation system, with the fulllength oleosin as a control, then immunoprecipitated using the oleosin antiserum (figure 3). The results show that although both the N-terminal (C115) and the C-terminal (N40) domains are efficiently immunoprecipitated by the antiserum (figure 3 C), the central hydrophobic domain of the oleosin is clearly not antigenic, since deletion CD was not immunoprecipitated (figure 3 A, B). Two other antisera raised against synthetic peptides based on the sequence of the central domain also failed to immunoprecipitate deletion CD (unpubl. obs.). We therefore concluded that it would not be possible to immunoprecipitate an oleosin protease-resistant fragment from the ‘run-on’ translated sunflower rER.
Figure 3. The central hydrophobic domain of the sunflower oleosin is not recognised by oleosin antisera. A, In vitro transcription/translation of full-length oleosin cDNA or template corresponding to the central hydrophobic domain. Translation products were analysed by SDS-PAGE and autoradiography. WTSO, Fulllength sunflower oleosin; CD, central domain only of sunflower oleosin. B, Immunoprecipitation of full-length and truncated oleosin translation products (as in A) was carried out. Only the WTSO (full-length protein) was immunoprecipitated, unlike the CD (central domain only) truncation. C, In vitro transcription/translation of either the full-length oleosin (WTSO), a N-terminal deletion (N40) or a C-terminal deletion (C115), followed by immunoprecipitation using the oleosin antisera indicated that both truncated forms, N40 and C115 (lacking respectively the entire N- and C-terminal domains), are efficiently precipitated. Some minor additional bands were obtained (*). These bands are likely to correspond to internal methionine codons being used as alternative start codons in the cell-free translation system.
2.5. In vitro synthesised oleosin inserts into sunflower smooth ER Another approach was therefore necessary to analyse the insertion of the oleosin into the sunflower microsomes. A coupled transcription/translation wheat germ cell-free extract was programmed with the fulllength sunflower oleosin cDNA, in the presence of EDTA-stripped-sunflower microsomal membranes. These ‘smooth’ microsomes had no associated ribosomal mRNAs (see figure 1, lane 2) and are therefore dependent on exogenous RNA (i.e. the in vitro transcribed sunflower oleosin transcript) templates. The translation products of the reaction were dual-labelled using L-[35S]-methionine and L-[3H]-leucine, and the activity of the sunflower microsomes was compared with that of canine pancreatic microsomes. After translation, the microsomes were isolated and incubated in the presence of proteinase K as previously described. This treatment generated a ∼8-kDa protected polypeptide, but only in the presence of sunflower or dog microsomes (figure 4 A, lanes 4, 6) vol. 37 (6) 1999
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Figure 4. N-terminal radio-sequencing of a membrane-insertedprotease resistant oleosin fragment. A, In vitro L-[35S]-methionineand L-[3H]-leucine-labelled transcription/translation of the full-length oleosin construct in the absence of (lanes 1, 2) or in the presence of (lanes 3, 4) ‘smooth’ sunflower microsomes, or canine microsomes (lanes 5, 6) was carried out, membranes purified by flotation, proteinase K-digested and carbonate-extracted. The resulting (protease-protected) polypeptides were identified by SDS-PAGE and fluorography. An 8-kDa protected fragment was obtained with both the sunflower and the canine microsomes (lanes 4, 6). The peptide marked with an arrow-head was transferred on a nitrocellulose membrane and analysed by N-terminal radio-sequencing. B, N-terminal radio-sequence derived from the Edman degradation (sixteen cycles) of the protease-resistant oleosin fragment. Each fraction was scintillation-counted for both [35S] and [3H] labels. No signal was obtained in the emission range for [35S] but four peaks were detected in the [3H] emission range. The corresponding position of these tritiated leucines (underlined) in the amino acid sequence of the sunflower oleosin [28] is also indicated.
whereas no protease-resistant polypeptide was observed in the absence of membranes (figure 4 A, lane 2). It is of interest to note that the protease-protected oleosin polypeptides generated by either sunflower or canine microsomes were of similar mobility, and that the predicted size of this polypeptide is very close to that observed for protease-treated oil bodies [14, 30].
2.6. Identification of the protease-protected oleosin domain by radio-sequencing Having confirmed that the sunflower oleosin was inserted into sunflower microsomal membranes in a carbonate- and protease-resistant manner, we wished Plant Physiol. Biochem.
to define the precise position of the protease-protected domain. Previously, Abell et al. [1] used a series of Nand C-terminal deletions to map the position of the protease-resistant domain of an Arabidopsis oleosin. Since this method is dependent on the resolution and sizing of small polypeptide fragments on polyacrylamide gels, we utilised a more precise approach. The (dual-labelled) in vitro synthesised protease-protected polypeptides were separated by polyacrylamide gel electrophoresis, blotted onto a nitrocellulose membrane and identified by autoradiography. The appropriate region of the membrane was then excised and subjected to automated Edman degradation radiosequencing [2]; sixteen cycles were performed and each cycle fraction was scintillation-counted for the presence of both [35S]- and [3H]-label. This showed that there was no detectable [35S]-methionine label within the sixteen cycles counted (corresponding to the first sixteen residues of the protease-protected portion of the oleosin). However, a number of signals were obtained from the [3H]-label as shown in figure 4 B. Four distinct peaks were identified, corresponding to the incorporation of [3H]-leucine into the oleosin protected fragment at positions 1, 7, 10 and 15. The amino acid sequence of the sunflower oleosin contains only one sequence which matches this pattern [LeuX5-Leu-X2-Leu-X4-Leu] which is just inside the start of the central hydrophobic domain. This region also lacks methionine residues, confirming the lack of recovered [35S]-methionine. Thus, the N-terminus of the protease-protected fragment of the sunflower oleosin can be mapped as starting at Leu50, as shown in figure 4 B. Although the protected fragment was derived from sunflower microsomal membranes, an identical pattern of incorporation was also observed with the protease-protected fragment from canine membranes (data not shown). This indicates that oleosin is likely to insert into the different microsomal membranes in a similar manner, and also validates the previous use of canine pancreatic microsomes for studies on the targeting of plant oleosins.
2.7. Comparison of oil body and microsomal membrane protease-protected oleosin fragments We have also recently determined the N-terminal sequence of the protected portion of the oleosin after protease-digestion of safflower oil bodies and found this to be Leu-Ala-Gly-Gly-Ser [14]. This motif is highly conserved among a number of dicot oleosins, such as those from Arabidopsis, rape, almond and citrus, and is found just inside the start of the central hydrophobic domain of the protein. Thus, it was
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Figure 5. Position of the N-terminal sequence of the protease-protected fragment within the sunflower oleosin. A comparison of the deduced amino acid sequences of the central domains of oleosins from sunflower, Arabidopsis, soybean, cotton, carrot and Brassica napus. The position of the start of the protease-protected fragment derived from microsomal insertion is indicated with a solid arrowhead and the position of the tritiated leucines identified by radio-sequencing indicated by dots. The start of the protease-protected fragment derived from oil bodies [14] is indicated with the open arrow, and the start of the hydrophobic domains indicated by the line.
possible to compare the N-termini of proteaseprotected fragments from oleosins inserted into microsomes or oil bodies. This showed (figure 5) that the protected fragments differ slightly in the position of their N-termini, with the sequence of the microsomal fragments starting one residue before that deduced for the oil body fragments. In addition, in both cases, the protease-resistant fragments do not start at the junction of the N-terminal domain and the central hydrophobic domain that has been predicted by modelling [11] or deletion analysis [1], but actually inside this central region. Importantly, it is also clear that the N-termini of these protease-protected fragments of the oleosin represent the start of the highly conserved central domain, as opposed to the start of the hydrophobic region per se. In the case of the sunflower oleosin, the hydrophobic region starts at Ile43 which is seven residues before the N-terminus of the protected fragment at Leu50. These data will therefore be useful in modelling the topology of the oleosin in either microsomal membranes or oil bodies.
3. CONCLUSION We provide evidence that the oleosin transcript is associated with the rough ER and that the oleosin protein is synthesised on bound ribosomes and cotranslationally inserted into the microsomal membrane. We have also determined the N-terminal sequence of a protease-protected fragment of microsomally inserted-oleosin, showing that not all of the central hydrophobic domain of the protein is inserted into the membrane. A similar sequence was observed for the oleosin in oil bodies.
4. METHODS 4.1. Plant material Sunflower plants (Helianthus annuus L., cv. Sunbred 246) were grown under greenhouse conditions with a photoperiod of 16 h light at 25 °C and 8 h dark at 18 °C. Seeds were harvested 15–17 d after flowering, corresponding to the mid-maturation stage. Chemicals were purchased from Sigma Chemicals (Poole, UK) unless otherwise stated. Radiochemicals were obtained from Amersham International (Amersham, UK). 4.2. Preparation of ER-derived microsomal membranes Embryonic tissue (1–2 g) was extracted from the developing seeds and homogenised on ice in 1–2 mL extraction buffer containing 0.6 M RNAse-free sucrose in 50 mM Hepes/KOH (pH 7.5), 10 mM KCl, 62.5 mM potassium acetate, 5 mM EGTA, 5 mM DTT and 2 units⋅mL–1 RNAsin (Promega) using a sterile mortar and pestle. Two variants of this buffer were used at this stage, buffer A supplemented with 5 mM MgCl2 and 1 % BSA (w/v) for extraction of rough microsomes or buffer B supplemented with 1 mM MgCl2, 5 mM EDTA and 1 % BSA (w/v) for extraction of smooth microsomes. The volume was increased to 12 mL with the appropriate extraction buffer and the cell homogenate was centrifuged at 1 000 × g for 15 min at 4 °C using a FO650 rotor (Beckman) to pellet the cell debris. The fat pad formed by the oil bodies was removed, the supernatant further centrifuged at 100 000 × g for 1 h at 4 °C in a TST 41.14 swing-out rotor (Kontron Instruments). The pellet was resuspended with 1 mL resuspension buffer C (identical to buffer A but containing 0.25 M sucrose), per g of tissue, and kept on ice until required. vol. 37 (6) 1999
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4.3. EDTA stripping of microsomes Microsomal membranes were extracted as described above with a buffer identical to buffer A but containing 100 µg⋅mL–1 PMSF instead of BSA. After the final 100 000 × g centrifugation, the microsomal pellet was resuspended in the same extraction buffer and the volume increased to 12 mL with a polyribosomereleasing buffer identical to buffer B but containing 100 µg⋅mL–1 PMSF instead of BSA and supplemented with 25 mM EDTA. This suspension was incubated 1 h at 4 °C with gentle mixing. The membranes were then pelleted again at 10 000 × g for 1 h at 4 °C as previously described. This washed membrane pellet was resuspended in 1 mL normal resuspension buffer C (BSA-containing) per g of tissue and kept on ice until required. The 100 000 × g supernatant from the washed microsomes was extracted with an equal volume of unbuffered phenol-chloroform and twice extracted with an equal volume of chloroform. The RNA was precipitated from the final supernatant with 2 M lithium acetate at –80 °C. After thawing on ice, the solution was centrifuged at 10 000 × g at 4 °C for 30 min using a JA-25 rotor (Beckman) and the pellet resuspended in 1 mL sterile water. The RNA was again precipitated with ethanol, desalted with 70 % ethanol and dissolved in water at a concentration of 1–2 mg⋅mL–1. This RNA stock was flash-frozen in liquid nitrogen and stored at –80 °C.
4.4. Oleosin truncated constructs A N-terminal deletion of an oleosin cDNA (pSO5; [28]) was generated by PCR using primer N40 (5’-GCCCATGGGCAAGATAATGGTCATCATGGCC3’) introducing a NcoI site (underlined), and the T7 terminator primer. A C-terminal deletion was generated similarly using primer C115 (5’-GCCTA ATACGACAACGAGCTTAACCC-3’) introducing a stop codon (underlined), and the T7 promoter primer. To allow a directional cloning, both fragments were digested with NcoI and ligated in a NcoI cut pCite T-vector system (Novagen) using a T4 DNA ligase (Promega, UK). The N-terminal truncation pN40 lacks the entire N-terminal amphipathic domain of the protein. The C-terminal truncation pC115 lacks the entire C-terminal hydrophilic domain. A construct lacking both the N- and C-terminal domains was produced using both primer N40 and C115 together and cloned as described. This N- and C-terminal truncation pCD corresponds to the central hydrophobic domain of the protein. Plant Physiol. Biochem.
4.5. In vivo translation using a wheat germ extract For translation of the microsome associated mRNAs, 50 µL reactions were carried out and the product was stored for up to 1 week at –20 °C. For each reaction, 5 µL membrane suspension or purified RNA was used as a template. In the case of released RNA, the stock solution was initially denatured by heating at 65 °C for 10 min. Then, 32.5 µL premix was added to the template and the reaction volume made up to 50 µL with sterile water. The premix was such that the final concentration of the reagents was 50 % wheat germ extract (Promega), 80 µM amino acid mixture (minus methionine), 0.8 U⋅µL–1 RNAsin and 1.85⋅107 Bq⋅mL–1 L-[35S]-methionine. These reactions were incubated for 1 h at 25 °C. In some cases, the translation was carried out using a centrifuged wheat germ extract in which microsomal membranes and free ribosomes were removed. For this purpose, 200 µL aliquots of wheat germ were centrifuged at 290 000 × g for 1 h at 4 °C in 8 × 34 mm polycarbonate tubes (Beckman) using a Beckman TLA100 rotor. Full-length and truncated clones of the oleosin cDNA were expressed in a cell-free coupled transcription/translation system (Promega) using the T7 RNA polymerase. Reactions were carried out in 10 µL following the manufacturer’s instructions and labelled in presence of 1.85⋅107 Bq⋅mL–1 L-[35S]methionine. After translation, the samples were either analysed further or mixed with an equal volume of 2× SDS-PAGE sample buffer. For the protease-protection assay, the full-length construct pSO5 was expressed in a coupled transcription/translation wheat germ extract but 20 µL reactions were translated in the presence or absence of either 2 µL canine microsomes or EDTAwashed sunflower microsomes. The products were labelled with 1⋅107 Bq⋅mL–1 L-[35S]-methionine and 1.85⋅107 Bq⋅mL–1 L-[3H]-leucine. After translation, the reaction volume was split in two microfuge tubes and one retained as non-digested control. In the other tube, proteinase K was added to give a final concentration of 200 µg⋅mL–1 (water instead of proteinase K was added in the control). The samples were then incubated for 30 min on ice and the reaction terminated by addition of PMSF to a final concentration of 2 mM and incubated for a further 5 min on ice. 4.6. Isolation and purification of the microsomes from the translation mixture After in vitro translation or proteinase K digestion, the samples were diluted with an equal volume of 0.5 M potassium acetate and the microsomes purified
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through a 0.6-M sucrose cushion containing 0.5 M potassium acetate, 5 mM MgCl2 and 50 mM HepesKOH (pH 7.9). The samples were centrifuged at 290 000 × g for 10 min at 4 °C using polycarbonate centrifuge tubes and a TLA-100 rotor as described previously. The microsomal pellets were resuspended in 50 µL buffer containing 0.25 M sucrose, 0.1 M potassium acetate, 5 mM MgCl2, 50 mM Hepes-KOH (pH 7.9) and sodium carbonate was added to a final concentration of 0.1 M (pH 11) [5]. The samples were incubated for 30 min on ice and the microsomes purified as described above.
4.7. Immunoprecipitation After translation or isolation and purification of the microsomes, the volume of each sample was increased to 500 µL with RIPA buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.1 % SDS (w/v), 1 % Nonidet P-40 (v/v), 0.5 % deoxycholic acid (w/v)) [13]. Oleosin polyclonal antiserum (3 µL) was added to each sample and incubated at 4 °C with gentle agitation for 1 h. Pre-swollen protein A-Sepharose (15 mg, Sigma) was added in each sample and the sample incubated overnight at 4 °C with gentle mixing. After binding, the beads were pelleted in a microcentrifuge, the supernatant removed, and the antigen/antibody/ protein-A complex gently washed by inversion with 500 µL RIPA buffer for 20 min at 4 °C. Several rounds of pelleting and washing in RIPA buffer were carried out, followed by a final wash with 10 mM Tris/HCl (pH 7.5), 0.1 % Nonidet P-40 (v/v). After centrifugation, the pellet was resuspended in 30 µL 2× SDSPAGE sample buffer and the mixture heated at 100 °C for 3 min to ensure the release of the protein from the protein A-Sepharose/IgG complex. When the supernatant from the initial incubation was retained for analysis, avoiding contaminations by the beads, the protein was precipitated with 10 % TCA for 30 min on ice. After centrifugation for 10 min at 13 000 × g, the pellets were washed twice with 1 mL 80 % acetone to remove the TCA and resuspended in 30 µL SDSPAGE sample buffer.
4.8. Proteinase protection assay After translation, in presence or absence of canine microsomes or EDTA-washed sunflower microsomes, the reaction volume was split in two microfuge tubes and one kept as non-digested control. In the other tube, proteinase K was added to give a final concentration of 200 µg⋅mL–1 (water instead of proteinase K was added in the control). The samples were then incubated on
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ice for 30 min and the reaction terminated by addition of PMSF to a final concentration of 2 mM and incubated for a further 5 min on ice.
4.9. Analysis by SDS-PAGE The proteins after either in vitro translation or immunoprecipitation were resolved by SDS-PAGE using a tricine buffer system based on that of Schagger and von Jagow [25]. The electrophoresis system consisted of a 15 and 4.5 % polyacrylamide in the separating gel and the stacking gel, respectively, and was run at 100 V until the loading dye ran off. After electrophoresis, the gels were fixed with water/ methanol/acetic acid (80/10/10 v/v/v), soaked in AmplifyTM (Amersham), dried and the products were detected by fluororadiography using BiomaxTM film (Kodak).
4.10. Radio-sequencing This was carried out essentially as described by Anderson and Gray [2]. N-terminal sequencing was carried out in the Department of Biochemistry, University of Bristol.
Acknowledgments IACR-Long Ashton Research Station receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the UK. DJL was the recipient of a BBSRC-ROPA award. FB was funded by the French Ministry of Higher Education and Research. We thank Dr W. Mawby (Department of Biochemistry, University of Bristol) for carrying out the radio-sequencing. Canine pancreatic microsomes were the kind gift of Dr Steve High, University of Manchester.
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