Xu H, Shields D
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, NY 10461.
Biochimie. 1994;76(3-4):257-64. doi: 10.1016/0300-9084(94)90155-4.
Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence has implicated either the trans-Golgi network (TGN) or immature secretory granules as the site of prohormone cleavage. To identify the site where prohormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin, proSRIF, (Stoller TJ, Shields D (1988) J Cell Biol 107, 2087-2095). By incubating these cells at 20 degrees C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident. Following the 20 degrees C block, the cells were permeabilized and proSRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35-50% efficient, required incubation at 37 degrees C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by pre-incubating the permeabilized cells with low concentrations of N-ethylmaleimide or bafilomycin, both inhibitors of vacuolar-type ATP-dependent proton pumps. These data suggest that ATP is required for generation of an acidic pH in the lumen of the TGN which is necessary for the activity of prohormone processing enzymes. By exploiting a permeabilized cell system, we have demonstrated that proSRIF cleavage is initiated in the TGN, in a reaction which is facilitated by a Golgi-associated vacuolar type ATPase.
许多肽类激素是以较大的前体形式合成的，这些前体在成对的碱性残基处进行内切蛋白水解切割，以产生生物活性分子。形态学证据表明，反式高尔基体网络（TGN）或未成熟分泌颗粒是激素原切割的部位。为了确定激素原切割起始的位点，我们使用了逆转录病毒感染的大鼠垂体前叶GH3细胞，这些细胞高水平表达前生长抑素（proSRIF）（Stoller TJ，Shields D（1988）J Cell Biol 107，2087 - 2095）。通过在20℃下培养这些细胞（该温度可阻止从高尔基体装置中排出），proSRIF定量积累在TGN中，且未观察到蛋白水解加工。在20℃阻断后，使细胞透化并测定proSRIF的加工情况。proSRIF切割成成熟激素的效率约为35% - 50%，需要在37℃下孵育并进行ATP水解，但与GTP或胞质溶胶无关。体外ATP依赖性的proSRIF加工受到弱碱氯喹、质子载体CCCP的抑制，或者通过用低浓度的N - 乙基马来酰亚胺或巴弗洛霉素（两者均为液泡型ATP依赖性质子泵抑制剂）预孵育透化细胞来抑制。这些数据表明，ATP是在TGN腔中产生酸性pH所必需的，而酸性pH对于激素原加工酶的活性是必要的。通过利用透化细胞系统，我们证明了proSRIF切割在TGN中起始，这一反应由高尔基体相关的液泡型ATP酶促进。
