Department of Developmental Genetics, Center for Plant Molecular Biology, University of Tübingen, Tübingen 72076, Germany.
BMC Plant Biol. 2012 Sep 12;12:164. doi: 10.1186/1471-2229-12-164.
In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport.
Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route.
Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.
在酵母和哺乳动物中，许多定位于降解的质膜（PM）蛋白都被泛素标记。这些泛素化的蛋白质被内吞到网格蛋白包被的小泡中，并被运输到早期内体隔室。在那里，通过内体分选复合物必需的运输（ESCRT）机制将泛素化的蛋白质分拣到多泡体的腔内小泡中。这些蛋白质在与溶酶体/溶酶体腔融合后降解，将其内容物释放到腔中。在植物中，已经发现一些在质膜和内体隔室之间循环的 PM 蛋白被泛素化，但尚不清楚泛素是否足以介导内化，因此是否作为内吞途径的主要分拣信号。为了测试植物是否将泛素作为膜蛋白降解的信号，我们将泛素翻译融合到不同的质膜荧光报告蛋白上，并分析它们的运输。
在烟草叶肉原生质体和烟草表皮细胞中，泛素标记的 PM 报告蛋白定位于内体和溶酶体腔中。如果通过共表达网格蛋白重链突变体（网格蛋白枢纽）抑制网格蛋白介导的内吞作用，这些报告蛋白的内化显著减少。令人惊讶的是，泛素标记的高尔基体报告蛋白也被运输到液泡腔中。如果抑制 ESCRT 机制，报告蛋白的液泡运输就会被阻断，这表明这些报告蛋白通过内吞运输途径被输送到液泡中。
泛素作为内质网系统不同隔室的分拣信号，将膜蛋白靶向到液泡降解途径中：如果在质膜上表达，泛素触发 PM 报告蛋白的内化进入内吞运输途径，但如果在高尔基体上表达，它也介导液泡的输送。在这两种情况下，泛素标记的蛋白质都通过早期内体和多泡体运输到溶酶体。这表明泛素化蛋白的液泡降解不仅限于质膜蛋白，还可能促进早期分泌途径中膜蛋白的周转。
