York Biomedical Research Institute, Department of Biology, University of York, York YO10 5DD, UK.
Imaging and Cytometry Laboratory, Bioscience Technology Facility, University of York, York YO10 5DD, UK.
Int J Mol Sci. 2021 Nov 19;22(22):12477. doi: 10.3390/ijms222212477.
Intracellular trafficking pathways control residency and bioactivity of integral membrane proteins at the cell surface. Upon internalisation, surface cargo proteins can be delivered back to the plasma membrane via endosomal recycling pathways. Recycling is thought to be controlled at the metabolic and transcriptional level, but such mechanisms are not fully understood. In yeast, recycling of surface proteins can be triggered by cargo deubiquitination and a series of molecular factors have been implicated in this trafficking. In this study, we follow up on the observation that many subunits of the Rpd3 lysine deacetylase complex are required for recycling. We validate ten Rpd3-complex subunits in recycling using two distinct assays and developed tools to quantify both. Fluorescently labelled Rpd3 localises to the nucleus and complements recycling defects, which we hypothesised were mediated by modulated expression of Rpd3 target gene(s). Bioinformatics implicated 32 candidates that function downstream of Rpd3, which were over-expressed and assessed for capacity to suppress recycling defects of ∆ cells. This effort yielded three hits: Sit4, Dit1 and Ldb7, which were validated with a lipid dye recycling assay. Additionally, the essential phosphatidylinositol-4-kinase Pik1 was shown to have a role in recycling. We propose recycling is governed by Rpd3 at the transcriptional level via multiple downstream target genes.
细胞内运输途径控制着整合膜蛋白在细胞表面的驻留和生物活性。在内化后，表面货物蛋白可以通过内体再循环途径返回质膜。再循环被认为受到代谢和转录水平的控制，但这些机制尚未完全理解。在酵母中，表面蛋白的再循环可以被货物去泛素化触发，并且一系列分子因子已被牵连到这种运输中。在这项研究中，我们跟进了许多 Rpd3 赖氨酸去乙酰化酶复合物亚基对于再循环是必需的这一观察结果。我们使用两种不同的测定法验证了十个 Rpd3 复合物亚基在再循环中的作用，并开发了定量这两种测定法的工具。荧光标记的 Rpd3 定位于细胞核并补充了再循环缺陷，我们假设这些缺陷是通过 Rpd3 靶基因(s)的调节表达介导的。生物信息学暗示 Rpd3 下游有 32 个功能候选者，我们对这些候选者进行了过表达，并评估了它们抑制 ∆ 细胞再循环缺陷的能力。这项工作产生了三个命中：Sit4、Dit1 和 Ldb7，并用脂质染料再循环测定法进行了验证。此外，必需的磷脂酰肌醇-4-激酶 Pik1 被证明在再循环中具有作用。我们提出，再循环是由 Rpd3 通过多个下游靶基因在转录水平上控制的。
