Secretion in yeast: preprotein binding to a membrane receptor and ATP-dependent translocation are sequential and separable events in vitro.
Secretion in yeast: preprotein binding to a membrane receptor and ATP-dependent translocation are sequential and separable events in vitro.
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DOI：
10.1083/jcb.108.6.2101
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发表时间：
1989-06
期刊：
影响因子：
--
通讯作者：
Meyer DI
中科院分区：
文献类型：
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作者：
Sanz P;Meyer DI
我们使用了一种无细胞质测定法，当亲和纯化的OmpA前体（proOmpA）用8 M尿素稀释到酵母粗粒体悬浊液中时，可以实现有效的易位和信号肽切割。这方面的蛋白质靶向和运输发生在两个可识别的步骤中：(a)在没有ATP和细胞质因子的情况下，前体与膜结合但不易位；(b) ATP的加入导致结合的前体易位并将其加工成成熟的形式。放射性标记的proOmpA与微粒体的结合是饱和的，并被未标记的proOmpA所抑制，而不是被成熟的OmpA或其他蛋白质所抑制。放射性标记的pre - α -因子的结合也会被其他前蛋白有效地竞争，但不会被成熟的前蛋白竞争。Scatchard分析显示proOmpA的Kd为7.5 X 10(-9) M.结合很可能是蛋白质介导的，因为用蛋白酶木瓜蛋白酶处理微粒体被发现具有抑制作用。这些结果代表了分泌蛋白前体结合膜的第一个功能表征。用NEM烷基化微粒体，用尿素洗涤膜或在非允许温度下使用（易位）突变体ptll的膜，不影响结合，但确实消除了随后的atp依赖性易位。将易位细分为单个反应的能力提供了一种更精确的方法来确定参与该过程的膜成分。
We have used a cytosol-free assay in which efficient translocation and signal peptide cleavage is achieved when the affinity-purified precursor of OmpA (proOmpA) is diluted out of 8 M urea into a suspension of yeast rough microsomes. This aspect of protein targeting and transport occurs in two discernible steps: (a) in the absence of ATP and cytosolic factors, the precursor binds to the membranes but is not translocated; (b) addition of ATP results in the translocation of the bound precursor and its processing to the mature form. The binding to microsomes of radiolabeled proOmpA is saturable and inhibited by the addition of unlabeled proOmpA but not by mature OmpA or other proteins. The binding of radiolabeled prepro-alpha-factor is also effectively competed by other preproteins, but not by mature ones. Scatchard analysis showed the Kd of proOmpA to be 7.5 X 10(-9) M. Binding is most likely protein mediated as treatment of the microsomes with the protease papain was found to be inhibitory. These results represent the first functional characterization of secretory protein precursor binding to membranes. Alkylation of the microsomes with NEM, washing the membranes with urea or using membranes from the (translocation) mutant ptll at the nonpermissive temperature, did not affect binding, but did eliminate the subsequent ATP-dependent translocation. The ability to subdivide translocation into individual reactions provides a more precise means of determining the membrane components involved in this process.