Direct purification of detergent-insoluble membranes from Medicago truncatularoot microsomes: comparison between floatation and sedimentation

BMC Plant Biology

Research Article

Sébastien Mongrand¹  Nicolas Deprêtre²  Daniel Wipf³  Christelle Guillier³  Françoise Simon-Plas³  Arnaud Mounier³  Ghislaine Recorbet³  Eliane Dumas-Gaudot³  Jean-Luc Cacas⁴ 

[1] CNRS, Laboratoire de Biogenèse Membranaire (LBM), Université Bordeaux UMR 5200, F-33000, Villenave d’Ornon, France;UMR CSGA: Centre des Sciences du Goût et de l’alimentation, UMR 6265 CNRS, 1324 INRA-uB, Dijon, France;UMR1347 INRA/Agrosup/Université de Bourgogne Agroécologie, Pôle Interactions Plantes-Microorganismes - ERL 6300 CNRS, BP 86510, 17 Rue Sully, F-21065, Dijon Cedex, France;UMR1347 INRA/Agrosup/Université de Bourgogne Agroécologie, Pôle Interactions Plantes-Microorganismes - ERL 6300 CNRS, BP 86510, 17 Rue Sully, F-21065, Dijon Cedex, France;CNRS, Laboratoire de Biogenèse Membranaire (LBM), Université Bordeaux UMR 5200, F-33000, Villenave d’Ornon, France;

关键词: Detergent insoluble membrane;    Proteomic;    Plant microdomain;    Microsomes;    Organelles;    Medicago truncatula;   

DOI  :  10.1186/s12870-014-0255-x

 received in 2014-05-27, accepted in 2014-09-20,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundMembrane 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.ResultsInferred 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.ConclusionsEven 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.

【 授权许可】

CC BY   
© Guillier et al.; licensee BioMed Central Ltd. 2014