Most cited article - PubMed ID 27622590
Enquiry into the Topology of Plasma Membrane-Localized PIN Auxin Transport Components
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.
Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors.
- Keywords
- PIN1, dimerization, hydrophilic hoop, intrinsic disorder, subcellular trafficking,
- MeSH
- Arabidopsis * metabolism MeSH
- Biological Transport MeSH
- Plant Roots metabolism MeSH
- Indoleacetic Acids metabolism MeSH
- Membrane Transport Proteins genetics metabolism MeSH
- Arabidopsis Proteins * genetics metabolism MeSH
- Intrinsically Disordered Proteins * genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Indoleacetic Acids MeSH
- Membrane Transport Proteins MeSH
- PIN1 protein, Arabidopsis MeSH Browser
- Arabidopsis Proteins * MeSH
- Intrinsically Disordered Proteins * MeSH
The proper distribution of the hormone auxin is essential for plant development. It is channeled by auxin efflux carriers of the PIN family, typically asymmetrically located on the plasma membrane (PM). Several studies demonstrated that some PIN transporters are also located at the endoplasmic reticulum (ER). From the PM-PINs, they differ in a shorter internal hydrophilic loop, which carries the most important structural features required for their subcellular localization, but their biological role is otherwise relatively poorly known. We discuss how ER-PINs take part in maintaining intracellular auxin homeostasis, possibly by modulating the internal levels of IAA; it seems that the exact identity of the metabolites downstream of ER-PINs is not entirely clear as well. We further review the current knowledge about their predicted structure, evolution and localization. Finally, we also summarize their role in plant development.
- Keywords
- ER-PINs, PIN proteins, PIN5, PIN8, auxin metabolism, auxin transport,
- Publication type
- Journal Article MeSH
- Review MeSH
The plant-specific proteins named PIN-FORMED (PIN) efflux carriers facilitate the direction of auxin flow and thus play a vital role in the establishment of local auxin maxima within plant tissues that subsequently guide plant ontogenesis. They are membrane integral proteins with two hydrophobic regions consisting of alpha-helices linked with a hydrophilic loop, which is usually longer for the plasma membrane-localized PINs. The hydrophilic loop harbors molecular cues important for the subcellular localization and thus auxin efflux function of those transporters. The three-dimensional structure of PIN has not been solved yet. However, there are scattered but substantial data concerning the functional characterization of amino acid strings that constitute these carriers. These sequences include motifs vital for vesicular trafficking, residues regulating membrane diffusion, cellular polar localization, and activity of PINs. Here, we summarize those bits of information striving to provide a reference to structural motifs that have been investigated experimentally hoping to stimulate the efforts toward unraveling of PIN structure-function connections.
- Keywords
- PIN efflux carriers, auxin transport, protein domains, sequence motifs, subcellular trafficking,
- Publication type
- Journal Article MeSH
- Review MeSH
The trafficking of subcellular cargos in eukaryotic cells crucially depends on vesicle budding, a process mediated by ARF-GEFs (ADP-ribosylation factor guanine nucleotide exchange factors). In plants, ARF-GEFs play essential roles in endocytosis, vacuolar trafficking, recycling, secretion, and polar trafficking. Moreover, they are important for plant development, mainly through controlling the polar subcellular localization of PIN-FORMED transporters of the plant hormone auxin. Here, using a chemical genetics screen in Arabidopsis thaliana, we identified Endosidin 4 (ES4), an inhibitor of eukaryotic ARF-GEFs. ES4 acts similarly to and synergistically with the established ARF-GEF inhibitor Brefeldin A and has broad effects on intracellular trafficking, including endocytosis, exocytosis, and vacuolar targeting. Additionally, Arabidopsis and yeast (Saccharomyces cerevisiae) mutants defective in ARF-GEF show altered sensitivity to ES4. ES4 interferes with the activation-based membrane association of the ARF1 GTPases, but not of their mutant variants that are activated independently of ARF-GEF activity. Biochemical approaches and docking simulations confirmed that ES4 specifically targets the SEC7 domain-containing ARF-GEFs. These observations collectively identify ES4 as a chemical tool enabling the study of ARF-GEF-mediated processes, including ARF-GEF-mediated plant development.
- MeSH
- Arabidopsis drug effects genetics metabolism MeSH
- Brefeldin A pharmacology MeSH
- Cell Membrane drug effects metabolism MeSH
- Chromones chemistry pharmacology MeSH
- DNA-Binding Proteins genetics metabolism MeSH
- Endocytosis drug effects MeSH
- Plants, Genetically Modified MeSH
- Membrane Glycoproteins genetics metabolism MeSH
- Membrane Transport Proteins genetics metabolism MeSH
- Mutation MeSH
- Protein Domains MeSH
- Arabidopsis Proteins genetics metabolism MeSH
- Saccharomyces cerevisiae Proteins genetics metabolism MeSH
- Saccharomyces cerevisiae drug effects metabolism MeSH
- Molecular Docking Simulation MeSH
- Transcription Factors genetics metabolism MeSH
- Protein Transport drug effects MeSH
- Guanine Nucleotide Exchange Factors chemistry genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- ARF1 protein, Arabidopsis MeSH Browser
- Brefeldin A MeSH
- Chromones MeSH
- DNA-Binding Proteins MeSH
- GNL1 protein, Arabidopsis MeSH Browser
- GNOM protein, Arabidopsis MeSH Browser
- Membrane Glycoproteins MeSH
- Membrane Transport Proteins MeSH
- PIN1 protein, Arabidopsis MeSH Browser
- Arabidopsis Proteins MeSH
- Saccharomyces cerevisiae Proteins MeSH
- SEC12 protein, S cerevisiae MeSH Browser
- Transcription Factors MeSH
- Guanine Nucleotide Exchange Factors MeSH
Clathrin-mediated endocytosis (CME) is a cellular trafficking process in which cargoes and lipids are internalized from the plasma membrane into vesicles coated with clathrin and adaptor proteins. CME is essential for many developmental and physiological processes in plants, but its underlying mechanism is not well characterized compared with that in yeast and animal systems. Here, we searched for new factors involved in CME in Arabidopsis thaliana by performing tandem affinity purification of proteins that interact with clathrin light chain, a principal component of the clathrin coat. Among the confirmed interactors, we found two putative homologs of the clathrin-coat uncoating factor auxilin previously described in non-plant systems. Overexpression of AUXILIN-LIKE1 and AUXILIN-LIKE2 in Arabidopsis caused an arrest of seedling growth and development. This was concomitant with inhibited endocytosis due to blocking of clathrin recruitment after the initial step of adaptor protein binding to the plasma membrane. By contrast, auxilin-like1/2 loss-of-function lines did not present endocytosis-related developmental or cellular phenotypes under normal growth conditions. This work contributes to the ongoing characterization of the endocytotic machinery in plants and provides a robust tool for conditionally and specifically interfering with CME in Arabidopsis.
- MeSH
- Arabidopsis genetics metabolism MeSH
- Endocytosis genetics physiology MeSH
- Clathrin genetics metabolism MeSH
- Arabidopsis Proteins genetics metabolism MeSH
- Seedlings genetics metabolism MeSH
- Protein Transport MeSH
- Protein Binding MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Clathrin MeSH
- Arabidopsis Proteins MeSH
Coordination of plant development requires modulation of growth responses that are under control of the phytohormone auxin. PIN-FORMED plasma membrane proteins, involved in intercellular transport of the growth regulator, are key to the transmission of such auxin signals and subject to multilevel surveillance mechanisms, including reversible post-translational modifications. Apart from well-studied PIN protein modifications, namely phosphorylation and ubiquitylation, no further post-translational modifications have been described so far. Here, we focused on root-specific Arabidopsis PIN2 and explored functional implications of two evolutionary conserved cysteines, by a combination of in silico and molecular approaches. PIN2 sequence alignments and modeling predictions indicated that both cysteines are facing the cytoplasm and therefore would be accessible to redox status-controlled modifications. Notably, mutant pin2C-A alleles retained functionality, demonstrated by their ability to almost completely rescue defects of a pin2 null allele, whereas high resolution analysis of pin2C-A localization revealed increased intracellular accumulation, and altered protein distribution within plasma membrane micro-domains. The observed effects of cysteine replacements on root growth and PIN2 localization are consistent with a model in which redox status-dependent cysteine modifications participate in the regulation of PIN2 mobility, thereby fine-tuning polar auxin transport.
- Keywords
- Arabidopsis, Auxin, PIN proteins, SRRF, intracellular distribution, plasma membrane protein sorting, protein mobility, protein modeling, root phenotype,
- MeSH
- Arabidopsis genetics metabolism MeSH
- Cysteine genetics MeSH
- Conserved Sequence * MeSH
- Plant Roots growth & development metabolism MeSH
- Indoleacetic Acids metabolism MeSH
- Membrane Microdomains metabolism MeSH
- Arabidopsis Proteins chemistry genetics metabolism MeSH
- Protein Transport MeSH
- Publication type
- Journal Article MeSH
- Names of Substances
- Cysteine MeSH
- Indoleacetic Acids MeSH
- PIN2 protein, Arabidopsis MeSH Browser
- Arabidopsis Proteins MeSH
