Kastner Christoph, Wagner Vera C, Fratini Marta, Dobritzsch Dirk, Fuszard Matthew, Heilmann Mareike, Heilmann Ingo
Department of Plant Biochemistry, Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, Charles-Tanford-Protein-Science-Center, Kurt-Mothes-Str. 3a, 06120, Halle (Saale), Germany.
Core Facility Proteomic Mass-Spectrometry, Martin Luther University Halle-Wittenberg, Charles-Tanford-Protein-Science-Center, Kurt-Mothes-Str. 3a, 06120, Halle (Saale), Germany.
Biochimie. 2022 Dec;203:65-76. doi: 10.1016/j.biochi.2022.10.002. Epub 2022 Oct 13.
Pollen tubes display polarized tip-growth and are a model to study the coordination of vesicular trafficking and cytoskeletal control. The molecular details of how dynamic actin filaments associate with the plasma membrane are currently unclear. In Arabidopsis thaliana, plasma membrane attachment of actin filaments may be mediated by four myosins representing the plant-specific myosin-subclass VIII, which localize to the plasma membrane and display only minor motor-activity. Here we explore the mode of membrane attachment of the pollen-expressed class VIII-myosins ATM2 and VIII-B through interaction with anionic membrane phospholipids. A fluorescent mCherry-ATM2-fusion decorated plasma membrane-peripheral actin filaments when expressed in tobacco pollen tubes, consistent with a role of class VIII-myosins at the membrane-cytoskeleton interface. As recombinant proteins, class VIII-myosins are prone to aggregation and to proteolysis, creating a challenge for their biochemical characterization. We describe a purification scheme for guanidinium chloride (GdmCl)-denatured recombinant proteins, followed by a renaturation protocol to obtain pure, soluble protein fragments of ATM2 and VIII-B. The fragments represent the C-terminal tail and coiled-coil-regions and lack the N-terminal actin-binding regions, IQ or motor domains. Based on lipid-overlays and liposome-sedimentation assays, the fragments of ATM2 and VIII-B bind anionic phospholipids. Small polybasic regions at the extreme C-termini were sufficient for lipid-binding of the respective protein fragments. When expressed in tobacco pollen tubes, a fluorescence-tagged variant of ATM2 lacking its lipid-binding region displayed substantially reduced plasma membrane association. The data indicate that class VIII-myosins may facilitate actin-plasma membrane attachment through interaction with anionic phospholipids, mediated by polybasic C-terminal lipid-binding domains.
花粉管表现出极性顶端生长，是研究囊泡运输协调和细胞骨架控制的模型。目前尚不清楚动态肌动蛋白丝如何与质膜结合的分子细节。在拟南芥中，肌动蛋白丝与质膜的附着可能由代表植物特异性肌球蛋白亚类VIII的四种肌球蛋白介导，它们定位于质膜且仅表现出轻微的运动活性。在这里，我们通过与阴离子膜磷脂相互作用来探索花粉表达的VIII类肌球蛋白ATM2和VIII-B的膜附着模式。当在烟草花粉管中表达时，荧光mCherry-ATM2融合蛋白装饰质膜周边的肌动蛋白丝，这与VIII类肌球蛋白在膜-细胞骨架界面的作用一致。作为重组蛋白，VIII类肌球蛋白容易聚集和被蛋白酶解，这给它们的生化特性表征带来了挑战。我们描述了一种用于氯化胍（GdmCl）变性重组蛋白的纯化方案，随后是复性方案，以获得ATM2和VIII-B的纯的、可溶的蛋白片段。这些片段代表C末端尾巴和卷曲螺旋区域，缺少N末端肌动蛋白结合区域、IQ或运动结构域。基于脂质覆盖和脂质体沉降分析，ATM2和VIII-B的片段结合阴离子磷脂。极端C末端的小多碱性区域足以实现各自蛋白片段的脂质结合。当在烟草花粉管中表达时，缺少脂质结合区域的ATM2荧光标记变体显示出质膜结合显著减少。数据表明，VIII类肌球蛋白可能通过由多碱性C末端脂质结合结构域介导的与阴离子磷脂的相互作用来促进肌动蛋白-质膜附着。
