Plant Cell Biology, Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, U.K.
Department of Animal and Plant Sciences, The University of Sheffield, Western Bank, Sheffield, U.K.
J Microsc. 2020 Nov;280(2):158-173. doi: 10.1111/jmi.12946. Epub 2020 Jul 31.
The plant Golgi apparatus is responsible for the processing of proteins received from the endoplasmic reticulum (ER) and their distribution to multiple destinations within the cell. Golgi matrix components, such as golgins, have been identified and suggested to function as putative tethering factors to mediate the physical connections between Golgi bodies and the ER network. Golgins are proteins anchored to the Golgi membrane by the C-terminus either through transmembrane domains or interaction with small regulatory GTPases. The golgin N-terminus contains long coiled-coil domains, which consist of a number of α-helices wrapped around each other to form a structure similar to a rope being made from several strands, reaching into the cytoplasm. In animal cells, golgins are also implicated in specific recognition of cargo at the Golgi.Here, we investigate the plant golgin Atgolgin-84A for its subcellular localization and potential role as a tethering factor at the ER-Golgi interface. For this, fluorescent fusions of Atgolgin-84A and an Atgolgin-84A truncation lacking the coiled-coil domains (Atgolgin-84AΔ1-557) were transiently expressed in tobacco leaf epidermal cells and imaged using high-resolution confocal microscopy. We show that Atgolgin-84A localizes to a pre-cis-Golgi compartment that is also labelled by one of the COPII proteins as well as by the tether protein AtCASP. Upon overexpression of Atgolgin-84A or its deletion mutant, transport between the ER and Golgi bodies is impaired and cargo proteins are redirected to the vacuole. LAY DESCRIPTION: The Golgi apparatus is a specialised compartment found in mammalian and plant cells. It is the post office of the cell and packages proteins into small membrane boxes for transport to their destination in the cell. The plant Golgi apparatus consist of many separate Golgi bodies and is responsible for the processing of proteins received from the endoplasmic reticulum (ER) and their distribution to multiple destinations within the cell. Specialised proteins called golgins have been suggested to tether Golgi bodies and the ER. Here we investigate the plant golgin Atgolgin-84A for its exact within the Golgi body and its potential role as a tethering factor at the ER-Golgi interface. For this, we have fused Atgolgin-84A with a fluorescent protein from jellyfish and we are producing this combination in tobacco leaf cells. This allows us to see the protein using laser microscopy. We show that Atgolgin-84A localises to a compartment between the ER and Golgi that is also labelled by the tether protein AtCASP. When Atgolgin-84A is produced in high amounts in the cell, transport between the ER and Golgi bodies is inhibited and proteins are redirected to the vacuole.
植物高尔基体负责处理从内质网（ER）接收的蛋白质，并将其分配到细胞内的多个目的地。已经鉴定出高尔基体基质成分，如 golgins，并提出它们作为假定的系留因子发挥作用，介导高尔基体和 ER 网络之间的物理连接。Golgins 通过跨膜结构域或与小调节 GTPase 的相互作用，通过 C 端锚定在高尔基体膜上。Golgin 的 N 端包含长的卷曲螺旋结构域，该结构域由多个α-螺旋组成，彼此缠绕形成类似于由几根股线组成的绳索的结构，延伸到细胞质中。在动物细胞中，golgins 也与高尔基体中货物的特异性识别有关。在这里，我们研究了植物 golgin Atgolgin-84A 的亚细胞定位及其作为 ER-Golgi 界面系留因子的潜在作用。为此，用烟草叶表皮细胞瞬时表达 Atgolgin-84A 的荧光融合物和缺乏卷曲螺旋结构域的 Atgolgin-84A 截断物（Atgolgin-84AΔ1-557），并使用高分辨率共焦显微镜进行成像。我们表明，Atgolgin-84A 定位于前顺面高尔基体隔室，该隔室也被 COPII 蛋白之一以及系留蛋白 AtCASP 标记。Atgolgin-84A 或其缺失突变体的过表达会损害 ER 和高尔基体之间的运输，并且货物蛋白被重定向到液泡。
