Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase
Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase
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DOI：
10.1111/j.1600-0854.2007.00616.x
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发表时间：
2007-10-01
期刊：
影响因子：
4.5
通讯作者：
Burd, Christopher G.
中科院分区：
文献类型：
--
作者：
Liu, Jingxuan;Sitaram, Anand;Burd, Christopher G.
酿酒酵母高亲和力铜转运蛋白Ctr1p介导细胞对Cu（I）的摄取。我们报道，当向缺铜细胞的生长培养基中添加铜（50μM CuSO₄）时，Ctr1p会迅速通过内吞作用被内化，被运送到类溶酶体的液泡腔中，并被液泡蛋白酶缓慢降解。通过对Ctr1p突变体的运输和降解分析，发现Ctr1p的C末端细胞质尾中的两个赖氨酸残基，Lys340和Lys345，对铜依赖的内吞作用和降解至关重要。在添加铜的情况下，发现Ctr1p被泛素化，并且Rsp5泛素连接酶的一个突变在很大程度上消除了泛素化、内吞作用和降解。在一个缺乏Rsp5p辅助因子Bul1p和Bul2p的菌株中，Ctr1p - 绿色荧光蛋白的内吞作用和降解显著减少。令人惊讶的是，一个缺乏Lys340和Lys345的Ctr1p突变体仍然以铜依赖的方式被泛素化，这表明Ctr1p在其他位点的泛素化不足以驱动铜依赖的内吞作用和降解。这项研究表明，铜通过刺激Rsp5p依赖的内吞作用以及Ctr1p在液泡中的降解来调节Ctr1p的周转。
The Saccharomyces cerevisiae high-affinity copper transporter, Ctr1p, mediates cellular uptake of Cu(I). We report that when copper (50 mu M CuSO4) is added to the growth medium of copper-starved cells, Ctr1p is rapidly internalized by endocytosis, delivered to the lumen of the lysosome-like vacuole and slowly degraded by vacuolar proteases. Through analysis of the trafficking and degradation of Ctr1p mutants, two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-green fluorescent protein were substantially diminished. Surprisingly, a Ctr1p mutant that lacks Lys340 and Lys345 was still ubiquitylated in a copper-dependent manner, indicating that ubiquitylation of Ctr1p on other sites is insufficient to drive copper-dependent endocytosis and degradation. This study demonstrates that copper regulates turnover of Ctr1p by stimulating Rsp5p-dependent endocytosis and degradation of Ctr1p in the vacuole.