Mayinger P, Meyer D I
Department of Biological Chemistry, UCLA School of Medicine.
EMBO J. 1993 Feb;12(2):659-66. doi: 10.1002/j.1460-2075.1993.tb05699.x.
The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 x 10(-5) M. ATP transport into ER vesicles was specifically inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3'-O-(4-benzoyl)benzoyl-ATP (Bz2-ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2-ATP blocked protein translocation in vitro measured co- as well as post-translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS-treated membranes. These results demonstrate that translocation requires a DIDS and Bz2-ATP-sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.
在酵母中，前体蛋白穿过糙面内质网（ER）膜的转运严格依赖于ATP的存在。由于转运需要Kar2p（哺乳动物BiP的酵母同源物），且它是一种ATP结合蛋白，因此ATP转运系统必定与ER的转运机制相偶联。我们在此报告酵母ER来源囊泡中ATP转运系统的特性。发现囊泡对ATP的摄取在微摩尔范围内具有饱和性，Km为1×10⁻⁵M。ATP转运到ER囊泡中会被4,4'-二异硫氰酸芪-2,2'-二磺酸（DIDS，一种已知可抑制许多其他阴离子转运蛋白的芪衍生物）和3'-O-(4-苯甲酰基)苯甲酰基-ATP（Bz2-ATP）特异性抑制。DIDS和Bz2-ATP对酵母微粒体中ATP摄取的抑制会阻断体外共翻译及翻译后测量的蛋白质转运。与ATP共同孵育可防止DIDS对转运的抑制作用。此外，选择性膜通透化使ATP能够进入内腔，恢复了DIDS处理膜的转运活性。这些结果表明，转运需要一个对DIDS和Bz2-ATP敏感的组分，其功能是将ATP转运到ER内腔。这些发现与酵母中蛋白质转运的当前模型一致，该模型规定Kar2p参与转运过程。
