Chimeric a-subunit isoforms generate functional yeast V-ATPases with altered regulatory properties in vitro and in vivo.
Chimeric a-subunit isoforms generate functional yeast V-ATPases with altered regulatory properties in vitro and in vivo.
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DOI：
10.1091/mbc.e22-07-0265
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发表时间：
2023-03-01
影响因子：
3.3
通讯作者：
Kane, Patricia M.
中科院分区：
文献类型：
--
作者：
Tuli, Farzana;Kane, Patricia M.
v - atp酶是高度调节的质子泵，使细胞器酸化。V-ATPase a亚基是一种双结构域蛋白，包含一个c端跨膜结构域负责质子运输和一个n端细胞质结构域（aNT），后者是一个调节枢纽，整合环境输入来调节V-ATPase的组装、定位和活性。酿酒酵母只编码两个细胞器特异性的a-异构体，高尔基体中的Stv1和液泡中的Vph1。在现有结构的基础上，我们设计了球状近端和远端互换的嵌合酵母蚂蚁。在体外，Vph1近端- stv1远端（VPSD）蚂蚁嵌合体与葡萄糖反应性RAVE组装因子结合，但与PI(3,5)P2的结合很少。Stv1近端- Vph1远端（SPVD） aNT缺乏RAVE结合，但与Vph1或Stv1相比，与磷酸肌苷的结合更紧密。当在体内连接到Vph1 c末端结构域时，两种嵌合体都能弥补Vph1∆突变体的生长缺陷，但只有SPVD嵌合体表现出野生型V-ATPase活性。含有SPVD嵌合体的细胞比野生型细胞更慢地适应低碳源，但在转向碱性后比野生型细胞生长得更快。这是“重新设计”的v - atp酶具有改变的调节特性和适应特定压力的第一个例子。
V-ATPases are highly regulated proton pumps that acidify organelles. The V-ATPase a-subunit is a two-domain protein containing a C-terminal transmembrane domain responsible for proton transport and an N-terminal cytosolic domain (aNT) that is a regulatory hub, integrating environmental inputs to regulate assembly, localization, and V-ATPase activity. The yeast Saccharomyces cerevisiae encodes only two organelle-specific a-isoforms, Stv1 in the Golgi and Vph1 in the vacuole. On the basis of recent structures, we designed chimeric yeast aNTs in which the globular proximal and distal ends are exchanged. The Vph1 proximal–Stv1 distal (VPSD) aNT chimera binds to the glucose-responsive RAVE assembly factor in vitro but exhibits little binding to PI(3,5)P2. The Stv1 proximal–Vph1 distal (SPVD) aNT lacks RAVE binding but binds more tightly to phosphoinositides than Vph1 or Stv1. When attached to the Vph1 C-terminal domain in vivo, both chimeras complement growth defects of a vph1∆ mutant, but only the SPVD chimera exhibits wild-type V-ATPase activity. Cells containing the SPVD chimera adapt more slowly to a poor carbon source than wild-type cells but grow more rapidly than wild-type cells after a shift to alkaline pH. This is the first example of a “redesigned” V-ATPase with altered regulatory properties and adaptation to specific stresses.