title: Direct purification of detergent-insoluble membranes from Medicago truncatula root microsomes: comparison between floatation and sedimentation.
abstract: BACKGROUND
  Membrane microdomains are defined as highly dynamic, sterol- and sphingolipid-enriched domains that resist to solubilization by non-ionic detergents. In plants, these so-called Detergent Insoluble Membrane (DIM) fractions have been isolated from plasma membrane by using conventional ultracentrifugation on density gradient (G). In animals, a rapid (R) protocol, based on sedimentation at low speed, which avoids the time-consuming sucrose gradient, has also been developed to recover DIMs from microsomes as starting material. In the current study, we sought to compare the ability of the Rapid protocol versus the Gradient one for isolating DIMs directly from microsomes of M. truncatula roots. For that purpose, Triton X-100 detergent-insoluble fractions recovered with the two methods were analyzed and compared for their sterol/sphingolipid content and proteome profiles.
  
  
  RESULTS
  Inferred from sterol enrichment, presence of typical sphingolipid long-chain bases from plants and canonical DIM protein markers, the possibility to prepare DIMs from M. truncatula root microsomes was confirmed both for the Rapid and Gradient protocols. Contrary to sphingolipids, the sterol and protein profiles of DIMs were found to depend on the method used. Namely, DIM fractions were differentially enriched in spinasterol and only shared 39% of common proteins as assessed by GeLC-MS/MS profiling. Quantitative analysis of protein indicated that each purification procedure generated a specific subset of DIM-enriched proteins from Medicago root microsomes. Remarkably, these two proteomes were found to display specific cellular localizations and biological functions. In silico analysis of membrane-associative features within R- and G-enriched proteins, relative to microsomes, showed that the most noticeable difference between the two proteomes corresponded to an increase in the proportion of predicted signal peptide-containing proteins after sedimentation (R) compared to its decrease after floatation (G), suggesting that secreted proteins likely contribute to the specificity of the R-DIM proteome.
  
  
  CONCLUSIONS
  Even though microsomes were used as initial material, we showed that the protein composition of the G-DIM fraction still mostly mirrored that of plasmalemma-originating DIMs conventionally retrieved by floatation. In parallel, the possibility to isolate by low speed sedimentation DIM fractions that seem to target the late secretory pathway supports the existence of plant microdomains in other organelles.
authors.affiliation: UMR1347 INRA/Agrosup/Université de Bourgogne Agroécologie, Pôle Interactions Plantes-Microorganismes - ERL 6300 CNRS, 17 Rue Sully, Dijon Cedex, BP 86510, F-21065, France. christelle.guillier@dijon.inra.fr.
authors.forename: Christelle
authors.initials: C
authors.lastname: Guillier
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Cacas
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Recorbet
authors.forename: Nicolas
authors.initials: N
authors.lastname: Deprêtre
authors.forename: Arnaud
authors.initials: A
authors.lastname: Mounier
authors.forename: Sébastien
authors.initials: S
authors.lastname: Mongrand
authors.forename: Françoise
authors.initials: F
authors.lastname: Simon-Plas
authors.forename: Daniel
authors.initials: D
authors.lastname: Wipf
authors.forename: Eliane
authors.initials: E
authors.lastname: Dumas-Gaudot
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry; D046913:Medicago truncatula; D021962:Membrane Microdomains / Q000737:chemistry; D008861:Microsomes; D018517:Plant Roots; D012995:Solubility
chemical_list: D003902:Detergents