KEX2-DEPENDENT INVERTASE SECRETION AS A TOOL TO STUDY THE TARGETING OF TRANSMEMBRANE PROTEINS WHICH ARE INVOLVED IN ER-]GOLGI TRANSPORT IN YEAST
KEX2-DEPENDENT INVERTASE SECRETION AS A TOOL TO STUDY THE TARGETING OF TRANSMEMBRANE PROTEINS WHICH ARE INVOLVED IN ER-]GOLGI TRANSPORT IN YEAST
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DOI：
10.1002/j.1460-2075.1994.tb06679.x
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发表时间：
1994-08-15
期刊：
影响因子：
11.4
通讯作者：
SCHMITT, HD
中科院分区：
文献类型：
--
作者：
BOEHM, J;ULRICH, HD;SCHMITT, HD
分离出的突变体在酵母早期分泌室中跨膜蛋白的保留方面存在缺陷。测试了一系列杂合蛋白在选择此类突变体中的用途。这些杂合蛋白均由 II 型跨膜蛋白（N-in/C-out 和转化酶 (Suc2) 作为报告分子，通过含有高尔基体蛋白酶 Kex2 切割位点的肽接头分隔开。所使用的整合膜蛋白（Sec12p、Sec22/Sly2p 或 Bet1/Sly12p）均已知是 ER-->酵母中的高尔基体运输。蔗糖酶很容易从含有 Sec22/Sly2p 或 Bet1/Sly12p 作为膜锚定部分的融合体中裂解下来。相反，表达 Sec12 转化酶的转化子需要两个不同基因中的任意一个发生突变才能分泌 Kex2 依赖性转化酶。显示出更强保留缺陷的突变体(rer1)被用来克隆相应的基因。 RER1 代表 III 号染色体着丝粒左侧的第一个阅读框。携带 RER1 基因破坏的细胞是可行的，并且表现出与原始突变体相同的错误定位表型。从核苷酸序列推断，Rer1 蛋白包含四个跨膜结构域。之前有人提出 Sec12p 在 ER 和顺式高尔基体之间循环。 使用 Sec12-转化酶和 rer1 突变体获得的一些结果类似于对哺乳动物细胞中高尔基驻留糖基转移酶和病毒蛋白保留的观察结果。例如，Sec12-转化酶的保留是不饱和的，并且 Sec12p 的跨膜结构域似乎构成了重要的靶向信号。
Mutants were isolated that are defective in the retention of a transmembrane protein in the early secretory compartments in yeast. A series of hybrid proteins was tested for their use in the selection of such mutants. Each of these hybrid proteins consisted of a type II transmembrane protein (N-in/C-out and invertase (Suc2) as a reporter separated by a peptide linker containing a cleavage site for the Golgi protease Kex2. The integral membrane proteins which were used-Sec12p, Sec22/Sly2p or Bet1/Sly12p-are all known to be required for ER-->Golgi transport in yeast. Invertase was readily cleaved from the fusions containing Sec22/Sly2p or Bet1/Sly12p as the membrane anchoring part. In contrast, Sec12-invertase expressing transformants required mutations in either of two different genes for Kex2-dependent invertase secretion. The mutant showing the stronger retention defect (rer1) was used to clone the corresponding gene. RER1 represents the first reading frame left of the centromere of chromosome III. Cells carrying a disruption of the RER1 gene are viable and show the same mislocalizing phenotype as the original mutants. The Rer1 protein, as deduced from the nucleotide sequence, contains four transmembrane domains. It has been suggested before that Sec12p cycles between the ER and the cis-Golgi compartment. Some results obtained by using Sec12-invertase and the rer1 mutants resemble observations on the retention of Golgi-resident glycosyltransferases and viral proteins in mammalian cells. For instance, retention of Sec12-invertase is non-saturable and the membrane-spanning domain of Sec12p seems to constitute an important targeting signal.