PURIFICATION AND INITIAL CHARACTERIZATION OF A POTENTIAL PLANT VACUOLAR TARGETING RECEPTOR
PURIFICATION AND INITIAL CHARACTERIZATION OF A POTENTIAL PLANT VACUOLAR TARGETING RECEPTOR
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DOI：
10.1073/pnas.91.8.3403
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发表时间：
1994-04-12
影响因子：
11.1
通讯作者：
ROGERS, JC
中科院分区：
文献类型：
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作者：
KIRSCH, T;PARIS, N;ROGERS, JC
已知网格蛋白包被的囊泡参与植物细胞中蛋白质从高尔基体到液泡的运输。蛋白质被引导进入该途径的机制尚不清楚。在这里，我们确定了一个完整的膜蛋白几乎等于80 kDa的，从发育豌豆（豌豆L.）子叶，其在中性pH下结合到用液泡硫醇蛋白酶原糊粉蛋白酶的N-末端靶向决定簇制备的亲和柱上，并且当pH降低至4时洗脱。该蛋白质未保留在用同源分泌型巯基蛋白酶内肽酶B的N-末端序列制备的对照柱上。80-kDa的蛋白质也积累在膜部分，这是密度小于网格蛋白包被的囊泡。体外研究表明，几乎等于80 kDa的蛋白质和糊粉蛋白原靶向决定簇之间的结合常数为37 nM。一个肽与空泡靶向决定簇从prosporamin弱竞争结合到几乎等于80 kDa的蛋白质，而一个肽携带一个单一的氨基酸取代已知废除prosporamin空泡靶向没有可测量的结合亲和力的蛋白质。结合蛋白是具有跨膜取向的糖蛋白，其中C末端暴露于细胞质。结合结构域位于蛋白质的N-末端管腔部分。结合蛋白的这些特性与受体的功能一致，该受体将选择反式高尔基体中的蛋白质以分选成网格蛋白包被的囊泡并递送至液泡。
Clathrin-coated vesicles are known to be involved in the transport of proteins from the Golgi to the vacuole in plant cells. The mechanisms by which proteins are directed into this pathway are not known. Here we identify an integral membrane protein of almost-equal-to 80 kDa, extracted from clathrin-coated vesicles of developing pea (Pisum sativum L.) cotyledons, that bound at neutral pH to an affinity column prepared with the N-terminal targeting determinant of the vacuolar thiol protease, proaleurain, and eluted when the pH was lowered to 4. The protein was not retained on a control column prepared with the N-terminal sequence of a homologous, secreted thiol protease, endopeptidase B. The 80-kDa protein also accumulated in a membrane fraction that is less dense than clathrin-coated vesicles. In vitro studies demonstrated a binding constant of 37 nM between the almost-equal-to 80-kDa protein and the proaleurain targeting determinant. A peptide with a vacuolar targeting determinant from prosporamin weakly competed for binding to the almost-equal-to 80-kDa protein, while a peptide carrying a single amino acid substitution known to abolish prosporamin vacuolar targeting had no measurable binding affinity for the protein. The binding protein is a glycoprotein with a transmembrane orientation in which the C terminus is exposed to the cytoplasm. The binding domain is located in the N-terminal luminal portion of the protein. These properties of the binding protein are consistent with the function of a receptor that would select proteins in the trans-Golgi for sorting to clathrin-coated vesicles and delivery to the vacuole.