Abstract
During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions.
Highlights
From the ‡Max Planck Institute of molecular Plant Physiology, Am Muhlenberg 1, 14476 Golm, Germany; §Department of Plant Systems Biology, University of Hohenheim, Garbenstrae 30, 70599 Stuttgart, Germany
Bootstrap Approach for Analysis of detergent resistant membrane fraction (DRM)/detergent soluble membrane fraction (DSF) Abundance Ratios—We developed a bootstrap based algorithm, named Unicorn, to statistically test differences between abundance ratios between DRM/DSF fractions in wild type compared with DRM/DSF abundance ratios on mcd treatment or in the sterol-biosynthesis mutant smt1
For proving reproducibility of the results obtained from the Unicorn algorithm, effects of mcd treatment and smt1 on protein DRM/DSF distribution were analyzed in 10 iterations
Summary
The abbreviations used are: DRM, detergent resistant membrane fraction; DSF, detergent soluble fraction; SP, soluble protein fraction; IM, intracellular membrane fraction; PPI, protein-protein interaction mcd methyl--cyclodextrin; FDR, false discovery rate; Lo, phase liquid ordered phase; Ld, phase liquid disordered phase. The total sterol composition in smt mutants was shown to be different from wild type, with the major phytosterols like sitosterol, stigmasterol, and brassicasterol being strongly depleted. Other sterol species remained unaltered and some even increased [20, 21] It remains unclear how the altered sterol-composition of the smt mutant affects sterol-protein interactions. In this study, using the newly developed algorithm Unicorn, we compared changes in protein distributions between DRM and DSF after biochemical mcd treatment and on endogenous alterations in sterol composition in smt to improve understanding of sterol–protein interactions
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
