The endoplasmic reticulum (ER) is an extensive network of intracellular membranes. Its major functions include proteosynthesis, protein folding, post-transcriptional modification and sorting of proteins within the cell, and lipid anabolism. Moreover, several studies have suggested that it may be involved in regulating intracellular auxin homeostasis in plants by modulating its metabolism. Therefore, to study auxin metabolome in the ER, it is necessary to obtain a highly enriched (ideally, pure) ER fraction. Isolation of the ER is challenging because its biochemical properties are very similar to those of other cellular endomembranes. Most published protocols for ER isolation use density gradient ultracentrifugation, despite its suboptimal resolving power. Here we present an optimised protocol for ER isolation from Arabidopsis thaliana seedlings for the subsequent mass spectrometric determination of ER-specific auxin metabolite profiles. Auxin metabolite analysis revealed highly elevated levels of active auxin form (IAA) within the ER compared to whole plants. Moreover, samples prepared using our optimised isolation ER protocol are amenable to analysis using various "omics" technologies including analyses of both macromolecular and low molecular weight compounds from the same sample.

• Klíčová slova
• auxin, density gradient centrifugation, endoplasmic reticulum, mass spectrometry, subcellular fractionation,
• MeSH
• Arabidopsis cytologie   metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• metabolom MeSH
• metabolomika metody   MeSH
• proteiny huseníčku analýza   metabolismus   MeSH
• proteomika metody   MeSH
• rostlinné buňky MeSH
• semenáček cytologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indoleacetic acid MeSH Prohlížeč
• kyseliny indoloctové MeSH
• proteiny huseníčku MeSH