Intracellular Transport and Secretion of Proteins in Plants

这项研究包括旨在了解大麦糊粉细胞内酶的运输和分泌的实验。由于其独特的性质，分离的糊粉原生质体已被开发用于蛋白质分泌的研究。大麦糊粉分离出的原生质体对赤霉酸和钙的反应与离体糊粉层的反应性质相似。利用原生质体，本实验室已经证明a-淀粉酶同工酶的低等电点(Low-PI)基团的前体形式被共价修饰为酶的分泌形式。这种修饰降低了同工酶等电点，而不显著影响蛋白质的相对分子质量。到目前为止的结果表明，这些同工酶没有被糖基化、磷酸化或硫酸盐化修饰。相反，这些数据表明了一种涉及侧链酰化的分泌蛋白质的翻译后修饰形式。来自ı14C！乙酸酯的放射性标记被特异性地结合到分泌形式的低等电点α-淀粉酶中。分泌蛋白质的前体没有标记，所以醋酸盐不会通过其代谢成氨基酸或糖而并入a-淀粉酶。高等电点α-淀粉酶的氨基末端乙酰化也被排除，因为这些蛋白质是末端封闭的。实验旨在研究α-淀粉酶酰化的精确性质和这种共价修饰的可能功能。利用非洲爪哇卵母细胞进行的实验也在进行中，这些实验提供了蛋白质酰化在分泌中的作用。大分子向细胞外分泌是高等动植物细胞的基本过程。当储存的食物储备在萌发过程中被调动时，这一过程在植物种子中尤其重要。我们对所涉及的酶的细胞内运输的了解是不完整的。这项研究解决了这一知识差距。*/

This research includes experiments aimed at understanding the intracellular transport and secretion of enzymes from aleurone cells of barley (Hordeum vulgare L. cv Himalaya). Because of their unique properties, isolated aleurone protoplasts have been exploited for studies of protein secretion. The response of protoplasts isolated from barley aleurone to gibberellic acid and calcium is qualitatively similar to that of isolated aleurone layers. Using protoplasts this lab has shown that precursor forms of the low isoelectric point (low-pI) group of a-amylase isozymes are covalently modified to the secreted forms of the enzyme. This modification results in the lowering of isozyme pI without significantly affecting the molecular weight of the protein. Results to date show that these isozymes are not modified by glycosylation, phosphorylation or sulfation. Rather the data indicate a form of posttranslational modification of secreted proteins involving side-chain acylation. Radiolabel from ı14C!acetate is specifically incorporated into secreted forms of low-pI a-amylase. The precursor of the secreted protein is not labeled, so acetate is not incorporated into a-amylase via its metabolism into amino acids or sugars. Amino-terminal acetylation of high-pI a-amylase is also ruled out since these proteins are end- blocked. Experiments are designed to investigate the precise nature of a-amylase acylation and the possible function of this covalent modification. Experiments with Xenopus oocytes that provide information on the role of protein acylation in secretion are also being conducted. The secretion of macromolecules to the cell exterior is a fundamental process of higher plant and animal cells. This process is particularly important in plant seeds when stored food reserves are mobilized during germination. Our knowledge of intracellular transport of the enzymes involved is incomplete. This research addresses this knowledge gap.***//