INTRACELLULAR EXPRESSION OF KLUYVEROMYCES-LACTIS TOXIN GAMMA-SUBUNIT MIMICS TREATMENT WITH EXOGENOUS TOXIN AND DISTINGUISHES 2 CLASSES OF TOXIN-RESISTANT MUTANT
INTRACELLULAR EXPRESSION OF KLUYVEROMYCES-LACTIS TOXIN GAMMA-SUBUNIT MIMICS TREATMENT WITH EXOGENOUS TOXIN AND DISTINGUISHES 2 CLASSES OF TOXIN-RESISTANT MUTANT
复制标题
DOI：
10.1002/yea.320070610
复制
发表时间：
1991-08-01
期刊：
影响因子：
2.6
通讯作者：
STARK, MJR
中科院分区：
文献类型：
--
作者：
BUTLER, AR;PORTER, M;STARK, MJR
乳酸菌毒素是一种异三聚体蛋白，在细胞周期的G1期不可逆地阻止敏感的酿酒酵母细胞的增殖。通过在敏感的酵母细胞中通过条件启动子表达毒素的γ亚基，先前已证明仅需要它才能抑制（Tokunaga et al.(1989)）。核酸学报17,3435-3446)。在这里，我们表明，与天然外源性毒素一样，细胞内γ -亚单位表达促进G1期敏感细胞的显著阻滞。然而，与天然毒素引起的G1阻滞不同，单独由γ -亚基诱导的G1阻滞不会导致细胞活力降低，并且是完全和快速可逆的，这表明G1阻滞和作用的不可逆性可能反映了毒素与敏感细胞相互作用的不同方面。为了更详细地研究其作用方式，我们选择了大量对该毒素具有高度抗性的酿酒葡萄球菌突变体。互补分析表明，除1个突变体外，其余突变体均为隐性突变，这些突变体定义了4个独立的基因。两个互补基团的成员同时获得对细胞内γ -亚基表达的抗性，表明它们含有修饰的毒素靶点。另外两个基因似乎是γ -亚基进入敏感细胞所必需的，因为这些突变体虽然对外源毒素不耐受，但对细胞内γ -亚基表达完全敏感。
The Kluyveromyces lactis toxin is a heterotrimeric protein which irreversibly arrests proliferation of sensitive Saccharomyces cerevisiae cells in the G1 phase of the cell cycle. By expressing the gamma-subunit of the toxin in sensitive yeast cells from a conditional promoter, it was previously demonstrated that it alone is required for inhibition (Tokunaga et al. (1989). Nucleic Acids Res. 17, 3435-3446). Here we show that, like native exogenous toxin, intracellular gamma-subunit expression promotes a striking arrest of sensitive cells in G1. However, unlike the G1 arrest caused by native toxin, that induced by the gamma-subunit alone does not result in reduced cellular viability and is fully and rapidly reversible, suggesting that the G1 arrest and the irreversibility of action may reflect different aspects of the toxin's interaction with sensitive cells. We have selected a large number of S. cerevisiae mutants which are highly resistant to the toxin in order to study its mode of action in more detail. Complementation analysis demonstrated that all but one of the mutants were recessive and these defined four separate genes. Members of two complementation groups concurrently acquired resistance to intracellular gamma-subunit expression, suggesting that they contain a modified toxin target site. The other two genes appear to be required for entry of the gamma-subunit into the sensitive cell since these mutants, while refractory to exogenous toxin, were fully sensitive to intracellular gamma-subunit expression.