Klionsky D J, Emr S D
California Institute of Technology, Pasadena 91125.
EMBO J. 1989 Aug;8(8):2241-50. doi: 10.1002/j.1460-2075.1989.tb08348.x.
The Saccharomyces cerevisiae PHO8 gene product, repressible alkaline phosphatase (ALP), is a glycoprotein enzyme that is localized to the yeast vacuole (lysosome). Using antibodies raised against synthetic peptides corresponding to two distinct hydrophilic sequences in ALP, we have been able to examine the biosynthesis, sorting and processing of this protein. ALP is synthesized as an inactive precursor containing a C-terminal propeptide that is cleaved from the protein in a PEP4-dependent manner. The precursor and mature protein are anchored in the membrane by an N-terminal hydrophobic domain that also appears to function as an uncleaved internal signal sequence. ALP has the topology of a type-II integral membrane protein containing a short basic N-terminal cytoplasmic tail that is accessible to exogenous protease when associated both with the endoplasmic reticulum and the vacuole. Similar to the soluble vacuolar hydrolases carboxypeptidase Y (CPY) and proteinase A (PrA), ALP transits through the early stages of the secretory pathway prior to vacuolar delivery. Two observations indicate, however, that ALP is localized to the vacuole by a mechanism which is in part different from that used by CPY and PrA: (i) maturation of proALP, which is indicative of vacuolar delivery, is less sensitive than CPY and PrA to the defects exhibited by certain of the vacuolar protein sorting (vps) mutants; and (ii) maturation of proALP proceeds normally in the presence of a potent vacuolar ATPase inhibitor, bafilomycin A1, which is known to block vacuole acidification and leads to the mis-sorting and secretion of precursor forms of CPY and PrA. These results indicate that ALP will be a useful model protein for studies of membrane protein sorting in yeast.
酿酒酵母PHO8基因产物，即可阻遏碱性磷酸酶（ALP），是一种糖蛋白酶，定位于酵母液泡（溶酶体）。利用针对与ALP中两个不同亲水区段相对应的合成肽产生的抗体，我们得以研究该蛋白的生物合成、分选和加工过程。ALP作为一种无活性前体合成，其含有一个C端前肽，该前肽以依赖PEP4的方式从蛋白上裂解下来。前体蛋白和成熟蛋白通过一个N端疏水结构域锚定在膜上，该结构域似乎也作为一个未裂解的内部信号序列发挥作用。ALP具有II型整合膜蛋白的拓扑结构，含有一个短的碱性N端胞质尾，当与内质网和液泡都相关联时，该尾可被外源蛋白酶作用。与可溶性液泡水解酶羧肽酶Y（CPY）和蛋白酶A（PrA）类似，ALP在液泡递送之前会经过分泌途径的早期阶段。然而，两项观察结果表明，ALP定位于液泡的机制部分不同于CPY和PrA所采用的机制：（i）前体ALP的成熟（这表明液泡递送）对某些液泡蛋白分选（vps）突变体所表现出的缺陷的敏感性低于CPY和PrA；（ii）前体ALP的成熟在强效液泡ATP酶抑制剂巴弗洛霉素A1存在的情况下正常进行，已知该抑制剂会阻断液泡酸化并导致CPY和PrA前体形式的错误分选和分泌。这些结果表明，ALP将是研究酵母中膜蛋白分选的有用模型蛋白。
