Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695.
Centre for Organismal Studies, Plant Developmental Biology, Heidelberg University, 69120 Heidelberg, Germany.
Proc Natl Acad Sci U S A. 2018 Aug 28;115(35):E8305-E8314. doi: 10.1073/pnas.1807763115. Epub 2018 Aug 13.
The vacuole is an essential organelle in plant cells, and its dynamic nature is important for plant growth and development. Homotypic membrane fusion is required for vacuole biogenesis, pollen germination, stomata opening, and gravity perception. Known components of the vacuole fusion machinery in eukaryotes include SNARE proteins, Rab GTPases, phosphoinositides, and the homotypic fusion and vacuolar protein sorting (HOPS) tethering complex. HOPS function is not well characterized in plants, but roles in embryogenesis and pollen tube elongation have been reported. Here, we show that HOPS subunits VPS33 and VPS41 accumulate in late endosomes and that VPS41, but not VPS33, accumulates in the tonoplast via a wortmannin-sensitive process. VPS41 and VPS33 proteins bind to liposomes, but this binding is inhibited by phosphatidylinosiltol-3-phosphate [PtdIns(3)P] and PtdIns(3,5)P, which implicates a nonconserved mechanism for HOPS recruitment in plants. Inducible knockdown of VPS41 resulted in dramatic vacuole fragmentation phenotypes and demonstrated a critical role for HOPS in vacuole fusion. Furthermore, we provide evidence for genetic interactions between VPS41 and VTI11 SNARE that regulate vacuole fusion, and the requirement of a functional SNARE complex for normal VPS41 and VPS33 localization. Finally, we provide evidence to support VPS33 and SYP22 at the initial stage for HOPS-SNARE interactions, which is similar to other eukaryotes. These results highlight both conserved and specific mechanisms for HOPS recruitment and function during vacuole fusion in plants.
液泡是植物细胞中的一种重要细胞器，其动态性质对植物的生长和发育至关重要。同源膜融合是液泡发生、花粉萌发、气孔开放和重力感应所必需的。真核生物液泡融合机制的已知成分包括 SNARE 蛋白、Rab GTPases、磷酸肌醇、同源融合和液泡分选（HOPS）连接复合物。HOPS 在植物中的功能尚未得到很好的描述，但已有报道称其在胚胎发生和花粉管伸长中发挥作用。在这里，我们表明 HOPS 亚基 VPS33 和 VPS41 聚集在晚期内体中，并且 VPS41（而不是 VPS33）通过wortmannin 敏感过程积累在液泡膜上。VPS41 和 VPS33 蛋白结合到脂质体上，但这种结合被磷脂酰肌醇-3-磷酸 [PtdIns(3)P] 和 PtdIns(3,5)P 抑制，这暗示了植物中 HOPS 募集的非保守机制。VPS41 的诱导性敲低导致液泡严重碎片化表型，并证明 HOPS 在液泡融合中起关键作用。此外，我们提供了证据表明 VPS41 和 VTI11 SNARE 之间存在遗传相互作用，调节液泡融合，并且功能 SNARE 复合物是 VPS41 和 VPS33 正常定位所必需的。最后，我们提供了证据支持 VPS33 和 SYP22 在 HOPS-SNARE 相互作用的初始阶段，这与其他真核生物相似。这些结果突出了 HOPS 在植物液泡融合过程中的募集和功能的保守和特异性机制。
