Andag U, Neumann T, Schmitt H D
Department of Molecular Genetics, Max-Planck-Institute for Biophysical Chemistry, D-37070 Göttingen, Germany.
J Biol Chem. 2001 Oct 19;276(42):39150-60. doi: 10.1074/jbc.M105833200. Epub 2001 Aug 7.
Sec22p is an endoplasmic reticulum (ER)-Golgi v-SNARE protein whose retrieval from the Golgi compartment to the endoplasmic reticulum (ER) is mediated by COPI vesicles. Whether Sec22p exhibits its primary role at the ER or the Golgi apparatus is still a matter of debate. To determine the role of Sec22p in intracellular transport more precisely, we performed a synthetic lethality screen. We isolated mutant yeast strains in which SEC22 gene function, which in a wild type strain background is non-essential for cell viability, has become essential. In this way a novel temperature-sensitive mutant allele, dsl1-22, of the essential gene DSL1 was obtained. The dsl1-22 mutation causes severe defects in Golgi-to-ER retrieval of ER-resident SNARE proteins and integral membrane proteins harboring a C-terminal KKXX retrieval motif, as well as of the soluble ER protein BiP/Kar2p, which utilizes the HDEL receptor, Erd2p, for its recycling to the ER. DSL1 interacts genetically with mutations that affect components of the Golgi-to-ER recycling machinery, namely sec20-1, tip20-5, and COPI-encoding genes. Furthermore, we demonstrate that Dsl1p is a peripheral membrane protein, which in vitro specifically binds to coatomer, the major component of the protein coat of COPI vesicles.
Sec22p是一种内质网(ER)-高尔基体v-SNARE蛋白,其从高尔基体区室向内质网(ER)的回收是由COPI囊泡介导的。Sec22p在ER还是高尔基体发挥其主要作用仍是一个有争议的问题。为了更精确地确定Sec22p在细胞内运输中的作用,我们进行了合成致死筛选。我们分离出了突变酵母菌株,其中SEC22基因功能(在野生型菌株背景下对细胞活力非必需)变得至关重要。通过这种方式,获得了必需基因DSL1的一个新的温度敏感突变等位基因dsl1-22。dsl1-22突变导致内质网驻留SNARE蛋白和带有C末端KKXX回收基序的整合膜蛋白从高尔基体向内质网的回收出现严重缺陷,以及利用HDEL受体Erd2p进行内质网再循环的可溶性内质网蛋白BiP/Kar2p的回收出现严重缺陷。DSL1与影响高尔基体到内质网再循环机制成分的突变在遗传上相互作用,即sec20-1、tip20-5和COPI编码基因。此外,我们证明Dsl1p是一种外周膜蛋白,其在体外特异性结合COPI囊泡蛋白衣的主要成分外套蛋白。