Pressure-induced endocytic degradation of the yeast low-affinity tryptophan permease Tat1 is mediated by Rsp5 ubiquitin ligase and functionally redundant PPxY-motif proteins
Pressure-induced endocytic degradation of the yeast low-affinity tryptophan permease Tat1 is mediated by Rsp5 ubiquitin ligase and functionally redundant PPxY-motif proteins
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压力诱导的酵母低亲和力色氨酸通透酶 Tat1 的内吞降解由 Rsp5 泛素连接酶和功能冗余 PPxY 基序蛋白介导
作者：
Suzuki;A.;Mochizuki;T.;Uemura;S.;Hiraki;T. and Abe;F.
酿酒酵母细胞表达两种色氨酸渗透酶，Tat 1和Tat 2，它们在对色氨酸的亲和力和细胞内定位方面具有不同的特征。虽然高亲和力通透酶Tat 2在其泛素依赖性降解方面已被充分证明，但低亲和力通透酶Tat 1尚未完全表征。在这里，我们表明，25 MPa的高静水压力触发Tat 1的降解，这取决于Rsp 5泛素连接酶和EH结构域的蛋白End 3。Tat 1是耐3-h放线菌酮处理，这表明它是高度稳定的正常生长条件下。Tat 1的泛素化最可能发生在N-末端赖氨酸29和31。精氨酸同时取代两个赖氨酸防止Tat 1降解，但单独取代它们中的任何一个都没有，这表明赖氨酸29和31的作用是多余的。当细胞暴露于高压时，Tat 1-GFP从质膜上完全丢失，而大量的Tat 1 K29 R-K31 R-GFP保留下来。HPG 1 -1（Rsp 5 P514 T）和rsp 5-ww 3突变使Tat 1在高压下稳定，但其中任何一个rsp 5-ww 1、rsp 5-ww 2和bul 1 Δ bul 2 Δ突变或编码抑制蛋白相关转运衔接子的基因的单一缺失都不能稳定Tat 1。然而，9-arrestins和Bul 1/Bul 2的同时损失阻止了Tat 1在25 MPa下的降解。结果表明，多个PPxY基序蛋白在调节Tat 1泛素化响应于高静水压力共享一些重要的作用。
Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1K29R-K31R-GFP remained. TheHPG1-1(Rsp5P514T) andrsp5-ww3mutations stabilized Tat1 under high pressure, but any one of thersp5-ww1,rsp5-ww2, andbul1Δbul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.