Seifu Yewubnesh Wendimu, Pukyšová Vendula, Rýdza Nikola, Bilanovičová Veronika, Zwiewka Marta, Sedláček Marek, Nodzyński Tomasz
Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, Brno, CZ-625 00, Czech Republic.
National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, Brno, CZ-625 00, Czech Republic.
Plant Methods. 2024 Jun 2;20(1):84. doi: 10.1186/s13007-024-01182-7.
PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.
PIN蛋白建立生长素浓度梯度，从而协调植物生长。PIN1 - 4和7定位于质膜（PM），促进生长素极性运输，而内质网（ER）定位的PIN5和PIN8维持细胞内生长素稳态。尽管已有报道称PIN5和PIN8蛋白在调节细胞内生长素稳态及其他发育事件中具有拮抗活性，但对这些蛋白的膜拓扑结构（这可能是其拮抗功能的基础）却知之甚少。在本研究中，我们优化了基于洋地黄皂苷的质膜通透方案，并结合免疫细胞化学标记来绘制拟南芥根细胞中PIN5和PIN8的膜拓扑结构。我们的结果表明，除了N端方向相似外，PIN5和PIN8的中央亲水环和C端方向相反，且跨膜结构域（TMD）数量不等。PIN8有十个TMD，由位于内质网腔的中央亲水环（HL）分隔成两组五个α螺旋，其N端和C端位于细胞质中。然而，PIN5的拓扑结构包含九个TMD。其N端和中央HL面向细胞质，而其C端位于内质网腔中。总体而言，本研究表明PIN5和PIN8蛋白具有不同的膜拓扑结构，同时引入了一套用于研究包括内质网膜定位蛋白在内的整合蛋白膜拓扑结构的方法工具包。
