Xu H, Shields D
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
J Cell Biol. 1993 Sep;122(6):1169-84. doi: 10.1083/jcb.122.6.1169.
Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence from several laboratories has implicated either the 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, T. J., and D. Shields. J. Cell Biol. 1988. 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; processing resumed upon shifting the cells back to 37 degrees C. After the 20 degrees C block, the cells were mechanically permeabilized and pro-SRIF 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 preincubating the permeabilized cells with low concentrations of N-ethylmaleimide, an inhibitor of vacuolar-type ATP-dependent proton pumps. These data suggest that: (a) proSRIF cleavage is initiated in the TGN, and (b) this reaction requires an acidic pH which is facilitated by a Golgi-associated vacuolar-type ATPase. A characteristic feature of polypeptide hormone-producing cells is their ability to store the mature hormone in dense core secretory granules. To investigate the mechanism of protein sorting to secretory granules, the budding of nascent secretory vesicles from the TGN was determined. No vesicle formation occurred at 20 degrees C; in contrast, at 37 degrees C, the budding of secretory vesicles was approximately 40% efficient and was dependent on ATP, GTP, and cytosolic factors. Vesicle formation was inhibited by GTP gamma S suggesting a role for GTP-binding proteins in this process. Vesicle budding was dependent on cytosolic factors that were tightly membrane associated and could be removed only by treating the permeabilized cells with high salt. After high salt treatment, vesicle formation was dependent on added cytosol or the dialyzed salt extract. The formation of nascent secretory vesicles contrasts with prosomatostatin processing which required only ATP for efficient cleavage. Our results demonstrate that prohormone cleavage which is initiated in the TGN, precedes vesicle formation and that processing can be uncoupled from the generation of nascent secretory vesicles.
许多肽类激素最初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，从而产生生物活性分子。来自多个实验室的形态学证据表明，反式高尔基体网络（TGN）或未成熟分泌颗粒是激素原切割的位点。为了确定激素原切割起始的位点，我们使用了经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）（斯托勒，T. J.，和D. 希尔兹。《细胞生物学杂志》。1988年。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 apparatus 中逸出），proSRIF 在 TGN 中定量积累，且未观察到明显的蛋白水解加工；当将细胞转回37℃时，加工过程恢复。在20℃阻断后，对细胞进行机械通透处理并测定 pro - SRIF 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 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（液泡型 ATP 依赖性质子泵的抑制剂）预孵育通透细胞来抑制。这些数据表明：（a）proSRIF 切割起始于 TGN，并且（b）该反应需要酸性 pH，这由高尔基体相关的液泡型 ATP 酶促进。产生多肽激素的细胞的一个特征是它们能够将成熟激素储存在致密核心分泌颗粒中。为了研究蛋白质分选到分泌颗粒的机制，我们确定了从 TGN 出芽形成新生分泌小泡的情况。在20℃时未发生小泡形成；相反，在37℃时，分泌小泡的出芽效率约为40%，并且依赖于 ATP、GTP 和胞质因子。GTPγS 抑制小泡形成，表明 GTP 结合蛋白在此过程中起作用。小泡出芽依赖于紧密结合在膜上的胞质因子，只有用高盐处理通透细胞才能将其去除。高盐处理后，小泡形成依赖于添加的胞质溶胶或透析后的盐提取物。新生分泌小泡的形成与前生长抑素加工形成对比，前生长抑素加工仅需要 ATP 进行有效切割。我们的结果表明，在 TGN 中起始的激素原切割先于小泡形成，并且加工过程可以与新生分泌小泡的产生解偶联。
