Direct binding of the Kex2p cytosolic tail to the VHS domain of yeast Gga2p facilitates TGN to prevacuolar compartment transport and is regulated by phosphorylation.
Direct binding of the Kex2p cytosolic tail to the VHS domain of yeast Gga2p facilitates TGN to prevacuolar compartment transport and is regulated by phosphorylation.
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DOI：
10.1091/mbc.e12-11-0843
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发表时间：
2013-02
影响因子：
3.3
通讯作者：
Fuller RS
中科院分区：
文献类型：
--
作者：
De M;Abazeed ME;Fuller RS
酵母 Gga1p 和 Gga2p 的 VHS 结构域结合 Kex2p 和 Vps10p 胞质尾部的位点 (GBS)。 Kex2p GBS 中 Ser-780 的磷酸化增强了 Kex2p 从 TGN 到 PVC 的转运，并且是由细胞壁损伤诱导的。 Kex2p GBS 功能通过直接结合、无细胞转运和 Kex2 定位的体内测定来显示。人类高尔基体定位的、含有 γ-耳的 ADP-核糖基化因子结合蛋白 (Ggas) 直接与细胞内受体的胞质尾部 (C 尾部) 中的酸性二亮氨酸分选基序结合。尽管有证据表明酵母 Ggas 在招募泛素化货物中发挥作用，但仍不清楚酵母 Ggas 是否也通过直接结合肽分选信号来发挥作用。双杂交分析表明，Gga1p 和 Gga2p Vps27、Hrs、Stam (VHS) 结构域均结合 Kex2p C 尾中的一个位点，Gga2p VHS 结构域结合 Vps10p C 尾中的一个位点。结合需要删除 Gga2p 铰链中明显的自抑制序列。 Kex2p C 尾中的 Ser780 对于结合至关重要：Ala 取代会阻断结合，但 Asp 取代会允许结合。使用纯化的 Gga2p VHS-GGA 和 TOM1 (GAT) 以及谷胱甘肽 S-转移酶-Kex2p C 尾融合物进行的生化测定表明，Gga2p 直接与 Kex2p C 尾结合，相对亲和力为 Asp780 > Ser780 > Ala780。针对含有磷酸-Ser780 的肽的亲和纯化抗体识别野生型 Kex2p，但不识别 S780A Kex2p，表明 Ser780 在体内被磷酸化； Ser780 的磷酸化会被细胞壁损伤药物上调。最后，Ser780 的突变改变了 Kex2p 在体内和无细胞跨高尔基体网络 (TGN)-液泡前区室 (PVC) 运输中的运输。因此，酵母 Gga 接头通过直接结合货物分子中非规范磷酸调节的 Gga 结合位点来促进 TGN-PVC 运输。
The VHS domains of yeast Gga1p and Gga2p bind sites (GBSs) in the Kex2p and Vps10p cytosolic tails. Phosphorylation of Ser-780 in the Kex2p GBS enhances Kex2p transport from the TGN to the PVC and is induced by cell wall damage. Kex2p GBS function is shown by direct binding, cell-free transport, and in vivo assays for Kex2 localization. Human Golgi-localized, γ-ear–containing, ADP-ribosylation factor–binding proteins (Ggas) bind directly to acidic dileucine sorting motifs in the cytosolic tails (C-tails) of intracellular receptors. Despite evidence for a role in recruiting ubiquitinated cargo, it remains unclear whether yeast Ggas also function by binding peptide-sorting signals directly. Two-hybrid analysis shows that the Gga1p and Gga2p Vps27, Hrs, Stam (VHS) domains both bind a site in the Kex2p C-tail and that the Gga2p VHS domain binds a site in the Vps10p C-tail. Binding requires deletion of an apparently autoinhibitory sequence in the Gga2p hinge. Ser780 in the Kex2p C-tail is crucial for binding: an Ala substitution blocks but an Asp substitution permits binding. Biochemical assays using purified Gga2p VHS–GGA and TOM1 (GAT) and glutathione S-transferase–Kex2p C-tail fusions show that Gga2p binds directly to the Kex2p C-tail, with relative affinities Asp780 > Ser780 > Ala780. Affinity-purified antibody against a peptide containing phospho-Ser780 recognizes wild-type Kex2p but not S780A Kex2p, showing that Ser780 is phosphorylated in vivo; phosphorylation of Ser780 is up-regulated by cell wall–damaging drugs. Finally, mutation of Ser780 alters trafficking of Kex2p both in vivo and in cell-free trans-Golgi network (TGN)–prevacuolar compartment (PVC) transport. Thus yeast Gga adaptors facilitate TGN–PVC transport by direct binding of noncanonical phosphoregulated Gga-binding sites in cargo molecules.