Plant retromer, localized to the prevacuolar compartment and microvesicles in Arabidopsis, may interact with vacuolar sorting receptors
Plant retromer, localized to the prevacuolar compartment and microvesicles in Arabidopsis, may interact with vacuolar sorting receptors
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DOI：
10.1105/tpc.105.035907
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发表时间：
2006-05-01
期刊：
影响因子：
11.6
通讯作者：
Robinson, DG
中科院分区：
文献类型：
--
作者：
Oliviusson, P;Heinzerling, O;Robinson, DG
在酵母和哺乳动物细胞中，在称为retromer的五聚体蛋白质复合物的帮助下，从中间的内体细胞器中检索出预定用于裂解室的酸性水解酶受体。我们克隆了拟南芥同源的三个酵母蛋白（Vps 35，Vps 29，和Vps 26）构成较大的亚基的retromer和制备抗血清。利用这些抗体，我们证明了从拟南芥微粒体制备的盐提取物中存在逆转录酶样蛋白复合物。该复合物与与液泡前区室标记物和高密度沉积膜共平衡的膜相关。免疫金阴性染色鉴定这些膜为90 nm直径的包被微泡。对烟草（烟草）BY-2细胞进行的共聚焦激光扫描免疫荧光研究显示，所有三种逆转录酶抗血清与前液泡室（PVC）标记物PEP 12和液泡分选受体VSRAt-1之间存在高度的共标记。植物retromer在多泡体的表面的存在下，也证明了从高压冷冻/冷冻替代标本获得的部分的免疫金标记。用渥曼青霉素处理BY-2细胞导致PVC肿胀和VPS 35和VSR信号分离。初步数据表明，retromer与VSR的胞质结构域相互作用，获得了通过免疫沉淀实验洗涤剂溶解微粒体与Vps 35抗体。
Receptors for acid hydrolases destined for the lytic compartment in yeast and mammalian cells are retrieved from intermediate, endosomal organelles with the help of a pentameric protein complex called the retromer. We cloned the Arabidopsis thaliana homologs of the three yeast proteins (Vps35, Vps29, and Vps26) constituting the larger subunit of retromer and prepared antisera against them. With these antibodies, we demonstrated the presence of a retromer-like protein complex in salt extracts prepared from Arabidopsis microsomes. This complex is associated with membranes that coequilibrate with prevacuolar compartment markers and with high-density sedimenting membranes. Immunogold negative staining identified these membranes as 90-nm-diameter coated microvesicles. Confocal laser scanning immunofluorescence studies performed on tobacco (Nicotiana tabacum) BY-2 cells revealed high degrees of colabeling between all three retromer antisera and the prevacuolar compartment (PVC) markers PEP12 and vacuolar sorting receptor VSRAt-1. The presence of plant retromer at the surface of multivesicular bodies was also demonstrated by immunogold labeling of sections obtained from high-pressure frozen/freeze-substituted specimens. Treatment of BY-2 cells with wortmannin led to swelling of the PVC and a separation of the VPS35 and VSR signals. Preliminary data suggesting that retromer interacts with the cytosolic domain of a VSR were obtained by immunoprecipitation experiments performed on detergent-solubilized microsomes with Vps35 antibodies.