The Npr1 kinase controls biosynthetic and endocytic sorting of the yeast Gap1 permease
The Npr1 kinase controls biosynthetic and endocytic sorting of the yeast Gap1 permease
复制标题
DOI：
10.1074/jbc.m102944200
复制
发表时间：
2001-11-23
影响因子：
4.8
通讯作者：
André, B
中科院分区：
文献类型：
--
作者：
De Craene, JO;Soetens, O;André, B
酿酒酵母总氨基酸渗透酶(Gap1)的膜转运受氮素调控。在以脯氨酸或尿素为唯一氮源生长的细胞中，新合成的Gap1被输送到质膜，在那里积累。在加入优先氮源NH4+后，GAPL被内吞并定位于液泡，在那里它被降解。这种下调需要渗透酶的泛素化，而这种泛素化依赖于必要的Npil/Rsp5泛素连接酶。在这项研究中，我们研究了Npr1激酶在调节Gap1运输中的作用。我们证明了Npr1是Gap1在质膜上稳定所必需的：当生长在Pro上的npr1(T8)突变体被转移到限制温度时，Gap1下调被触发，就像当NH4+加入野生型细胞时一样。新合成的Gap1在进入质膜的途中也受到Npr1的控制：在npr1 Delta突变体中，新合成的Gap1从高尔基体分离到液泡中，而不通过质膜。在生长在NH4+上的野生型细胞中，观察到新合成的Gap1与液泡类似的直接分选。最后，Gap I在NPR1细胞中被磷酸化，但这种磷酸化并不严格依赖于Npr1。我们的结果表明，Npr1激酶在Gap1转运的生理控制中起着核心作用，这种控制不仅作用于存在于质膜上的Gap1，也作用于分泌途径的晚期Gap1。Npr1属于蛋白激酶的一个亚类，其中一些被报道对其他通透性的活性起正向调控作用。我们认为这些激酶也可以作为渗透酶运输的调节因子。
Membrane trafficking of the general amino acid permease (Gap1) of Saccharomyces cerevisiae is under nitrogen regulation. In cells growing on proline or urea as the sole nitrogen source, newly synthesized Gap1 is delivered to the plasma membrane, where it accumulates. Upon addition of NH4+, a preferential nitrogen source, Gapl is endocy-tosed and targeted to the vacuole, where it is degraded. This down-regulation requires ubiquitination of the permease, and this ubiquitination is dependent on the essential Npil/Rsp5 ubiquitin ligase. In this study, we investigated the role of the Npr1 kinase in the regulation of Gap1 trafficking. We show that Npr1 is required for stabilization of Gap1 at the plasma membrane: when an npr1(t8) mutant growing on proline is shifted to the restrictive temperature, Gap1 down-regulation is triggered, as it is when NH4+ is added to wild-type cells. The fate of newly synthesized Gap1 en route to the plasma membrane is also under Npr1 control: in an npr1 Delta mutant, neosynthesized Gap1 is sorted from the Golgi to the vacuole without passing via the plasma membrane. Similar direct sorting of neosynthesized Gap1 to the vacuole was observed in wild-type cells grown on NH4+. Finally, Gap I is phosphorylated in NPR1 cells, but this phosphorylation is not strictly dependent on Npr1. Our results show that Npr1 kinase plays a central role in the physiological control of Gap1 trafficking and that this control is exerted not only on Gap1 present at the plasma membrane but also on Gap1 late in the secretory pathway. Npr1 belongs to a subgroup of protein kinases, some of which are reported to exert a positive control on the activity of other permeases. We propose that these kinases also function as regulators of permease trafficking.