Steel G J, Harley C, Boyd A, Morgan A
The Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, United Kingdom.
Mol Biol Cell. 2000 Apr;11(4):1345-56. doi: 10.1091/mbc.11.4.1345.
An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum-Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex of N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure-function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulated SEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. The sec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr(394)-->Pro). Thr(394) is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast alpha-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts.
一种进化上古老的机制被用于细胞内膜融合事件，其范围涵盖从酵母内质网到高尔基体的运输，再到人类大脑中突触小泡的胞吐作用。该机制的核心是由N - 乙基马来酰亚胺敏感融合蛋白（NSF）、可溶性NSF附着蛋白（SNAPs）和SNAP受体（SNAREs）组成的核心复合体。尽管这些蛋白质被公认为囊泡运输中的关键参与者，但其分子作用机制仍不清楚。为了阐明NSF中重要的结构 - 功能关系，开展了一项针对酵母NSF（Sec18p）显性负突变体的筛选。这涉及对受GAL1调控的SEC18酵母表达质粒进行随机诱变。基于半乳糖诱导的生长停滞鉴定出了几个显性负等位基因，其中一个sec18 - 109被详细表征。sec18 - 109的表型（通过生物合成途径的异常膜运输、膜性管状网络的积累、生长抑制、细胞密度增加）是由于SEC18中单个A - G替换导致Sec18p中的错义突变（Thr(394)→Pro）。Thr(394)在大多数AAA蛋白中保守，实际上构成了最小AAA共有序列的一部分，该共有序列是这个大蛋白家族的一个标志。对重组Sec18 - 109p的分析表明，该突变并不阻止六聚体形成或与酵母α - SNAP（Sec17p）的相互作用，但会导致无法检测到的ATP酶活性，且该活性不能被Sec17p刺激。这表明AAA蛋白共有序列在调节ATP水解中起作用。此外，这种在酵母中筛选显性负突变体的方法可应用于其他保守蛋白，以突出其哺乳动物对应物中的重要功能域。
