Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain; Centre for Mechanochemical Cell Biology, Gibbet Hill Road, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK(1).
Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas CSIC, Ramiro de Maeztu 9, Madrid 28040, Spain.
Fungal Genet Biol. 2019 Feb;123:78-86. doi: 10.1016/j.fgb.2018.12.003. Epub 2018 Dec 11.
Coatomer-I (COPI) is a heteromeric protein coat that facilitates the budding of membranous carriers mediating Golgi-to-ER and intra-Golgi transport. While the structural features of COPI have been thoroughly investigated, its physiological role is insufficiently understood. Here we exploit the amenability of A. nidulans for studying intracellular traffic, taking up previous studies by Breakspear et al. (2007) with the α-COP/CopA subunit of COPI. Endogenously tagged α-COP/CopA largely localizes to SedV syntaxin-containing early Golgi cisterna, and acute inactivation of ER-to-Golgi traffic delocalizes COPI to a haze, consistent with the cisternal maturation model. In contrast, the Golgi localization of COPI is independent of the TGN regulators HypB and HypA, implying that COPI budding predominates at the SedV early Golgi, with lesser contribution of the TGN. This finding agrees with the proposed role of COPI-mediated intra-Golgi retrograde traffic in driving cisternal maturation, which predicts that the capacity of the TGN to generate COPI carriers is low. The COPI early Golgi compartments intimately associates with Sec13-containing ER exit sites. Characterization of the heat-sensitive copA1ts (sodC1) mutation showed that it results in a single residue substitution in the ε-COP-binding Carboxyl-Terminal-Domain of α-COP that likely destabilizes its folding. However, we show that Golgi disorganization by copA1ts necessitates >150 min-long incubation at 42 °C. This weak subcellular phenotype makes it unsuitable for inactivating COPI traffic acutely for microscopy studies, and explains the aneuploidy-stabilizing role of the mutation at subrestrictive temperatures.
衣被体-I（COPI）是一种异源蛋白衣被，它促进介导高尔基体到内质网和内高尔基运输的膜载体的出芽。虽然 COPI 的结构特征已经得到了彻底的研究，但它的生理作用还没有被充分理解。在这里，我们利用构巢曲霉易于研究细胞内运输的特点，借鉴了 Breakspear 等人之前的研究（2007 年），使用 COPI 的 α-COP/CopA 亚基。内源性标记的 α-COP/CopA 主要定位于 SedV 含有 syntaxin 的早期高尔基 cisterna，急性内质网到高尔基体运输失活会使 COPI 解聚到一个晕圈中，这与 cisterna 成熟模型一致。相比之下，COPI 的高尔基体定位不依赖于 TGN 调节剂 HypB 和 HypA，这意味着 COPI 出芽主要发生在 SedV 早期高尔基体，而 TGN 的贡献较小。这一发现与 COPI 介导的内高尔基逆行运输在推动 cisterna 成熟中的作用一致，这表明 TGN 产生 COPI 载体的能力较低。COPI 早期高尔基体 compartment 与含有 Sec13 的内质网出口位点密切相关。copA1ts（sodC1）突变的热敏感特性的表征表明，它导致 α-COP 的 ε-COP 结合羧基末端结构域中的单个残基取代，这可能使其折叠不稳定。然而，我们表明，copA1ts 引起的高尔基体紊乱需要在 42°C 下孵育 >150 分钟。这种弱亚细胞表型使其不适合用于在显微镜研究中急性失活 COPI 运输，并且解释了该突变在亚限制温度下稳定非整倍体的作用。
