De Fea K A, Nakahara D H, Calayag M C, Yost C S, Mirels L F, Prusiner S B, Lingappa V R
Department of Physiology, University of California, San Francisco 94143-0444.
J Biol Chem. 1994 Jun 17;269(24):16810-20.
The prion protein (PrP) displays some unusual features in its biogenesis. In cell-free systems it can be synthesized as either an integral transmembrane protein spanning the membrane twice, with both amino and carboxyl domains in the lumen of the endoplasmic reticulum, or as a fully translocated polypeptide. A charged, extracytoplasmic region, termed the Stop Transfer Effector (STE) sequence, has been shown to direct the nascent translocating chain to stop at the adjoining hydrophobic domain to generate the first membrane-spanning region (TM1). However, the determinants of the second translocation event in the biogenesis of the transmembrane form have not been identified previously. Moreover, the relationship of transmembrane and fully translocated forms of PrP has not been well understood. Here, we report progress in resolving both of these issues. Using protein chimeras in cell-free translation systems and Xenopus oocytes, we identify the sequence which directs nascent PrP to span the membrane a second time, with its carboxyl-terminal domain in the endoplasmic reticulum lumen. Surprisingly, PrP carboxyl-terminal domain translocation does not appear to be directed by an internal signal or signal-anchor sequence located downstream of TM1, as would have been expected from studies of other multispanning membrane proteins. Rather, carboxyl-terminal domain translocation appears to be another consequence of the action of STE-TM1, that is, the same sequence responsible for generating the first membrane-spanning region. Studies of an STE-TM1-containing protein chimera in Xenopus oocytes demonstrate that most of these chains upon completion of their translation, initially span the membrane twice, with a topology similar to that of transmembrane PrP, but are carbonate-extractable. These chains have the transmembrane orientation only transiently and chase into a fully translocated form. These results support a model in which a metastable "transmembrane" intermediate, residing within the aqueous environment of the translocation channel, can be converted into either the integrated transmembrane or the fully translocated form of PrP, perhaps directed by trans-acting factor (s). Such a model may explain why stable the transmembrane isoform of PrP has not been observed in normal cells and how nascent PrP might be directed to alternate pathways of folding.
朊病毒蛋白（PrP）在其生物合成过程中表现出一些不同寻常的特征。在无细胞系统中，它可以被合成为一种跨膜两次的整合跨膜蛋白，其氨基和羧基结构域都在内质网腔中，也可以被合成为一种完全转运的多肽。一个带电荷的胞外区域，称为终止转移效应器（STE）序列，已被证明能引导新生的转运链在相邻的疏水结构域处停止，从而产生第一个跨膜区域（TM1）。然而，此前尚未确定跨膜形式生物合成过程中第二次转运事件的决定因素。此外，PrP的跨膜形式和完全转运形式之间的关系也尚未得到很好的理解。在此，我们报告在解决这两个问题方面所取得的进展。通过在无细胞翻译系统和非洲爪蟾卵母细胞中使用蛋白质嵌合体，我们确定了引导新生PrP第二次跨膜的序列，其羧基末端结构域位于内质网腔中。令人惊讶的是，PrP羧基末端结构域的转运似乎不是由位于TM1下游的内部信号或信号锚定序列所引导的，而这是根据对其他多次跨膜蛋白的研究本应预期到的。相反，羧基末端结构域的转运似乎是STE-TM1作用的另一个结果，也就是说，是负责产生第一个跨膜区域的相同序列。对非洲爪蟾卵母细胞中一种含有STE-TM1的蛋白质嵌合体的研究表明，这些链中的大多数在翻译完成后，最初跨膜两次，其拓扑结构与跨膜PrP相似，但可被碳酸盐提取。这些链只是短暂地具有跨膜方向，随后转变为完全转运的形式。这些结果支持了一个模型，即存在于转运通道水环境中的一种亚稳态“跨膜”中间体，可以被转化为PrP的整合跨膜形式或完全转运形式，这可能是由反式作用因子所引导的。这样一个模型或许可以解释为什么在正常细胞中未观察到稳定的PrP跨膜异构体，以及新生PrP可能是如何被引导至不同的折叠途径的。
