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Cloning, Characterization and in Vitro Targeting of Two Unique Proteins in Sesame Oil Bodies
植物種子特有的胞器─油體，它儲存三酸甘油酯(triacylglycerols)，提供種子發芽及發芽後生長所需之能量來源，目前所提出的油體構造模型是三酸甘油酯包覆於單層磷脂質(phospholipid)內，磷脂質表面則鑲滿了油體膜蛋白(oleosin)以及一些微量蛋白質。油體膜蛋白為一鹼性蛋白質，分子量約在15-26 kDa之間，它提供了負電荷斥力以及立體障礙，使得油體在種子內能以互相擠壓的形式充滿整個細胞，即使在長時間的儲存之後，仍然保有其胞器之穩定性。油體膜蛋白依其分子量與蛋白結構，區分為H、L兩種異構型態，觀察芝麻油體蛋白在Tricine SDS-PAGE的分析結果，發現除了兩個特有的油體膜蛋白(17, 15 kDa)之外，還存在另一油體膜蛋白(15.5 kDa)，參考大豆油體膜蛋白(24 kDa)的核酸序列篩選得到芝麻油體膜蛋白(15.5 kDa)的cDNA，與其他油體膜蛋白進行胺基酸序列比較，發現此蛋白質應是目前已知的最小分子量、H型態油體膜蛋白，並推測蛋白質結構中C-端高保留區amphipathic a-helix，可能具有特別的生理活性。
三個命名為Sop1, 2, 3的芝麻油體微量蛋白質，藉由胺基酸序列分析的結果設計degenerate 引子進行PCR反應，將得到的核酸片段藉由E. coil進行蛋白表現，再以西方墨點法證實其為油體微量蛋白Sop1，而且在其他種子油體內亦發現存在著與Sop1相似的蛋白質。Sop1蛋白與Ca2+離子反應後，蛋白質在SDS-PAGE電泳位移的速度較快，推測Sop1蛋白是一個Ca2+離子結合性蛋白質，因此將它命名為油體鈣蛋白(caleosin)。依蛋白質結構與胺基酸親、疏水性，將油體鈣蛋白區分為三個區域 : N端親水性Ca2+離子結合區、中間疏水性與C端親水性磷酸化反應區。比較油體鈣蛋白、油體膜蛋白的蛋白質中間疏水性胺基酸序列，發現兩者均具有高保留性Proline knot motif，猜測此區域可能與油體蛋白標的傳送到油體表面有關。
為了進一步了解油體蛋白如何運送及定位分布於油體表面上，我們建立一個細胞外的標的分析系統，將oleosin、caleosin在兔子網狀紅血球抽出物轉譯系統和TAG、PLs經超音波均質化得到的乳狀物進行反應。由實驗的結果推測油體蛋白應是自發性標的到油體膜上，過程中並無其他因子參與。比較乳狀物與微粒體在轉錄反應中，油體蛋白oleosin 、caleosin傾向標的到油體乳狀物上。以中性的PLs如PC、PE合成的乳狀物，我們發現油體蛋白標的效率降低，如將帶負電荷的PLs如PS、PI提高到組成比例的20%，油體蛋白標的效率則相近於以自然PLs組成比例的人造油體。另一方面，以突變的caleosin蛋白，檢視蛋白結構對油體蛋白標的到油體的重要性，推測caleosin結構中與oleosin相似的Proline knot motif，應扮演一個決定性的角色。
The oil bodies of plant seeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with alkaline proteins termed oleosins. Two distinct oleosins are present in the oil bodies of diverse angiosperms, and classified as high and low Mr isoforms according to their relative molecular masses in each species. In sesame oil bodies, besides the two ubiquitous oleosin isoforms (17 and 15 kDa), an additional minor oleosin (15.5 kDa) was revealed on Tricine SDS-PAGE. A full-length cDNA fragment was cloned, sequenced and deduced to be a putative oleosin of 15,446 Da. The gene was constructed in a fusion or non-fusion vector and then over-expressed with different efficiency in E. coli. All three oleosins purified from sesame oil bodies were subjected to immuno-assaying using antibodies raised against the over-expressed oleosin. The results confirmed that this gene encodes the sesame 15.5 kDa oleosin. Sequence comparisons with other known oleosins revealed that sesame 15.5 kDa oleosin does not represent a new oleosin isoform class but may have been derived through gene duplication and truncation of sesame 17 kDa oleosin, and possesses the minimal structure of the high Mr oleosin isoform. A conserved amphipathic a-helix is predicted in sesame 15.5 kDa oleosin, which may imply a potential biological function associated with this isoform.
Plant seed oil bodies comprise a matrix of triacylglycerols surrounded by a monolayer of phospholipids embedded with abundant oleosins and some minor proteins. Three minor proteins, temporarily termed Sops 1-3, have been identified in sesame oil bodies. A cDNA sequence of Sop1 was obtained by PCR cloning using degenerate primers derived from two partial amino acid sequences, and subsequently confirmed via immunological recognition of its over-expressed protein in Escherichia coli. Alignment with four published homologous sequences suggests Sop1 as a putative calcium-binding protein. Immunological cross-recognition implies that this protein, tentatively named caleosin, exists in diverse seed oil bodies. Caleosin migrated faster in SDS-PAGE when incubated with Ca2+. Ca2+A single copy of caleosin gene was found in sesame genome based on Southern hybridization. Northern hybridization revealed that both caleosin and oleosin genes were concurrently transcribed in maturing seeds where oil bodies are actively assembled. Hydropathy plot and secondary structure analysis suggest that caleosin comprises three structural domains, i.e., an N-terminal hydrophilic calcium-binding domain, a central hydrophobic anchoring domain, and a C-terminal hydrophilic phosphorylation domain. Compared with oleosin, a conserved proline knot-like motif is located in the central hydrophobic domain of caleosin and assumed to involve in protein assembly onto oil bodies.
An in vitro system was established to examine protein targeting to maturing seed oil bodies by incubating oleosin or caleosin translated in a reticulocyte lysate system with artificial oil emulsions composed of triacylglycerol and phospholipid. The results suggest that oil body proteins spontaneously target to maturing oil bodies without apparent assistance of other factors in a co-translational mode. Translated caleosin and oleosin preferred to target to artificial oil emulsions instead of microsomal membranes. Low targeting efficiency was detected when the phospholipid of artificial oil emulsions comprised neutral phosphatidylcholine or phosphatidylethanolamine. The targeting efficiency could be substantially elevated when 20% negatively charged phosphatidylserine and/or phosphatidylinositol was supplemented to phosphatidylcholine in artificial oil emulsions to mimic the native phospholipid composition of oil bodies. Mutated caleosin lacking various structural domains or subdomains was examined for its in vitro targeting efficiency. The results indicate that the subdomain comprising the proline knot motif plays a crucial role in caleosin targeting or assembling to oil bodies. A model describing direct targeting of oil-body proteins to maturing oil bodies was proposed.
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