Kirsch T, Paris N, Butler J M, Beevers L, Rogers J C
Department of Botany and Microbiology, University of Oklahoma, Norman 73019.
Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3403-7. doi: 10.1073/pnas.91.8.3403.
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 approximately 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 approximately 80 kDa protein and the proaleurain targeting determinant. A peptide with a vacuolar targeting determinant from prosporamin weakly competed for binding to the approximately-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.
已知网格蛋白包被小泡参与植物细胞中蛋白质从高尔基体到液泡的运输。蛋白质被导向该途径的机制尚不清楚。在这里，我们从发育中的豌豆（Pisum sativum L.）子叶的网格蛋白包被小泡中提取了一种约80 kDa的整合膜蛋白，该蛋白在中性pH下与用液泡硫醇蛋白酶原aleurain的N端靶向决定簇制备的亲和柱结合，并在pH降至4时洗脱。该蛋白不保留在用同源分泌硫醇蛋白酶内肽酶B的N端序列制备的对照柱上。80 kDa的蛋白也积累在一个比网格蛋白包被小泡密度小的膜组分中。体外研究表明，约80 kDa的蛋白与原aleurain靶向决定簇之间的结合常数为37 nM。来自前孢子蛋白的具有液泡靶向决定簇的肽对与约80 kDa蛋白的结合有微弱竞争，而携带已知可消除前孢子蛋白液泡靶向的单个氨基酸取代的肽对该蛋白没有可测量的结合亲和力。结合蛋白是一种糖蛋白，具有跨膜方向，其C末端暴露于细胞质。结合结构域位于蛋白的N端腔内部分。结合蛋白的这些特性与一种受体的功能一致，该受体可在反式高尔基体中选择蛋白质，以便分选到网格蛋白包被小泡并输送到液泡。
