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| Content Provider | Springer Nature : BioMed Central |
|---|---|
| Author | Seifu, Yewubnesh Wendimu Pukyšová, Vendula Rýdza, Nikola Bilanovičová, Veronika Zwiewka, Marta Sedláček, Marek Nodzyński, Tomasz |
| Abstract | 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. |
| Related Links | https://plantmethods.biomedcentral.com/counter/pdf/10.1186/s13007-024-01182-7.pdf |
| Ending Page | 16 |
| Page Count | 16 |
| Starting Page | 1 |
| File Format | HTM / HTML |
| ISSN | 17464811 |
| DOI | 10.1186/s13007-024-01182-7 |
| Journal | Plant Methods |
| Issue Number | 1 |
| Volume Number | 20 |
| Language | English |
| Publisher | BioMed Central |
| Publisher Date | 2024-06-02 |
| Access Restriction | Open |
| Subject Keyword | Plant Sciences Biological Techniques Arabidopsis thaliana Auxin homeostasis and transport Digitonin PM-permeabilizing protocols Endoplasmic reticulum Immunocytochemistry Membrane topology determination methods PIN auxin efflux carriers |
| Content Type | Text |
| Resource Type | Article |
| Subject | Plant Science Biotechnology Genetics |
| Journal Impact Factor | 4.7/2023 |
| 5-Year Journal Impact Factor | 5.6/2023 |
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