Endosomal proteases facilitate the fusion of endosomes with vacuoles at the final step of the endocytotic pathway.
Endosomal proteases facilitate the fusion of endosomes with vacuoles at the final step of the endocytotic pathway.
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DOI：
10.1111/j.1365-313x.2005.02349.x
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发表时间：
2005-02
期刊：
影响因子：
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通讯作者：
Kenji Yamada;Kentaro Fuji;T. Shimada;M. Nishimura;I. Hara-Nishimura
中科院分区：
文献类型：
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作者：
Kenji Yamada;Kentaro Fuji;T. Shimada;M. Nishimura;I. Hara-Nishimura
质膜蛋白质被运输到液泡中降解的机制在植物中还没有得到很好的表征。为了阐明质膜蛋白是如何降解的，我们用荧光内吞标记物FM 4 -64监测了烟草悬浮培养的BY-2细胞中的内吞途径。由于核内体能快速有效地传递到液泡中，因此几乎检测不到核内体。有趣的是，我们发现木瓜蛋白酶家族蛋白酶抑制剂E-64 d在蔗糖饥饿条件下引起细胞内大量内体的积累。该结果表明E-64 d减弱了内体与空泡的融合。我们确定了两个木瓜蛋白酶同系物，这是本地化的内体，与生物素化的抑制剂。我们将其命名为内体定位木瓜蛋白酶（ENPs）。免疫荧光分析显示，液泡分选受体，前液泡室（PVC）的标志物，是本地化的内涵体。该结果及其酸性性质表明，内体对应于PVC。这些结果表明，ENPs促进在蔗糖饥饿条件下的液泡运输途径的最后一步。我们进一步研究了E-64 d对组成型表达由绿色荧光蛋白（GFP）和质膜蛋白（GFP-PIP 2a或GFP-LTI 6 b）组成的融合蛋白的两种转基因拟南芥植物的影响。在这些转基因植物的根细胞质膜上观察到GFP荧光。用E-64 d处理诱导GFP-荧光内体的积累并抑制这些融合蛋白的降解。在E-64 d处理的转基因植物的液泡中没有观察到GFP荧光。两者合计，这些结果表明，内体蛋白酶的融合所需的内体与液泡在最后一步的细胞内吞途径降解质膜蛋白在植物中。
The mechanism by which plasma membrane proteins are transported to vacuoles for degradation has not been well characterized in plants. To clarify how plasma membrane proteins are degraded, we monitored the endocytotic pathway in tobacco suspension-cultured BY-2 cells with a fluorescent endocytosis marker, FM4-64. Because of the efficient and rapid delivery of endosomes to the vacuoles, endosomes were scarcely detectable. Interestingly, we found that E-64d, an inhibitor of papain family proteases, caused the accumulation of a large number of endosomes in the cells under the sucrose-starved condition. This result indicates that E-64d attenuates the fusion of endosomes with vacuoles. We identified two papain homologues, which are localized in the endosomes, with a biotinylated inhibitor. We designated them as endosome-localized papains (ENPs). Immunofluorescent analysis revealed that vacuolar sorting receptor, a marker of prevacuolar compartment (PVC), was localized in the endosomes. This result and their acidic nature show that the endosomes correspond to PVC. These results suggest that ENPs facilitate the final step in the vacuolar trafficking pathway under the sucrose-starved condition. We further examined the effects of E-64d on two transgenic Arabidopsis plants that constitutively express a fusion protein composed of green fluorescent protein (GFP) and a plasma membrane protein (GFP-PIP2a or GFP-LTI6b). GFP fluorescence was observed on the plasma membrane of root cells in these transgenic plants. Treatment with E-64d induced the accumulation of GFP-fluorescent endosomes and inhibited the degradation of these fusion proteins. No GFP fluorescence was observed in vacuoles in E-64d-treated transgenic plants. Taken together, these results suggest that endosomal proteases are required for the fusion of endosomes with vacuoles at the final step in the endocytotic pathway for degradation of plasma membrane proteins in plants.