Abell B M, Holbrook L A, Abenes M, Murphy D J, Hills M J, Moloney M M
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Plant Cell. 1997 Aug;9(8):1481-93. doi: 10.1105/tpc.9.8.1481.
An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N- and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-beta-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-beta-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.
以拟南芥油质蛋白为模型研究油质蛋白的拓扑结构及其向油体的靶向运输。油质蛋白mRNA在体外与犬微粒体一起以一系列截短形式进行翻译。这使得能够通过蛋白酶K图谱分析膜拓扑结构。油质蛋白维持一种构象，其膜整合疏水结构域两侧是位于微粒体表面外侧的N端和C端结构域。这是内质网（ER）上一种独特的膜拓扑结构。油质蛋白疏水结构域“脯氨酸结”基序内的三个普遍保守的脯氨酸残基被亮氨酸残基取代。体外翻译后，仅观察到蛋白酶K保护方面的微小差异。这些差异在大豆微粒体中不明显。两种油质蛋白形式在ER上的整合效率没有显著差异。然而，作为油质蛋白-β-葡萄糖醛酸酶翻译融合体，脯氨酸结变体在瞬时胚胎表达和稳定转化种子中均未能靶向油体。对表达油质蛋白-β-葡萄糖醛酸酶融合体的转基因胚胎进行分级分离显示，脯氨酸结变体与天然形式相比在ER中积累到相似水平。因此，脯氨酸结基序对于ER整合和拓扑结构的确定并不重要，但对于油体靶向是必需的。脯氨酸结的缺失导致油质蛋白多肽在运输过程中内在的不稳定性。
