Unraveling Sterol-dependent Membrane Phenotypes by Analysis of Protein Abundance-ratio Distributions in Different Membrane Fractions Under Biochemical and Endogenous Sterol Depletion
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Authors: | Henrik Zauber Witold Szymanski Waltraud X. Schulze |
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Affiliation: | From the ‡Max Planck Institute of molecular Plant Physiology, Am Mühlenberg 1, 14476 Golm, Germany; ;§Department of Plant Systems Biology, University of Hohenheim, Garbenstraβe 30, 70599 Stuttgart, Germany |
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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.The plasma membrane incorporates a broad spectrum of proteins covering mainly different structural, signaling or transport functionalities. Being the first semipermeable cell barrier to its surrounding environment the plasma membrane is important for metabolite transport as well as initiation point of several signaling processes (1–4). To maintain cell homeostasis, protein activity as well as complex formation through protein protein interactions (PPI) need to be tightly regulated. The major regulating mechanisms are postranslational modification of proteins and modulated abundances of proteins present in the plasma membrane. Another potential regulating mechanism became apparent with the discovery of sterol and sphingolipid enriched domains (microdomains) in the plasma membrane (5–8, 3). Microdomain like structures have been shown to form spontaneously in artificial plasma membranes (9). After a decade of research on these structures, microdomains turned out to be particularly involved in signaling and transport processes incorporating a specific set of proteins. Microdomains provide subcompartments in the plasma membrane with specific physicochemical properties that on specific sterol protein interactions might alter protein activity or PPIs. With the discovery of microdomains the fluid lipid mosaic model was extended by distinguishing two plasma membrane phases, an ordered phase of lower density (Lo phase) enriched in sterols, sphingolipids and long chain fatty acids and a disordered phase of higher density (Ld phase). From isolated plasma membranes a lower density and a higher density membrane fraction can be separated in a sucrose gradient after treatment with non-ionic detergents. The resulting detergent resistant membrane fraction (DRM)1 is related to Lo phase and high density detergent soluble membrane fraction (DSF) relates to Lo phases. Although it is still under debate how well DRMs represent native plasma membrane microdomains (10–12), research on protein-sterol interactions is possible by usage of sterol depleting agents like methyl-β-cyclodextrin mβcd (13). Therefore mβcd is suitable for detecting false positive cholesterol protein interactions in DRM studies (14–19). Proteins depleted on mβcd treatment are finally considered to be sterol dependent (15–17). To compare the mβcd treatment for disturbing the sterol distribution in the Lo fraction, we studied the sterol biosynthesis deficient mutant smt1. (20) smt1 carries a point mutation in the smt1 locus, encoding the sterol methyltransferase 1 and it exhibits a dwarf-like phenotype on whole plant level (20). In total, three sterol methyl transferases are encoded in Arabidopsis where SMT1 catalyzes the first step in the sterol biosynthesis by adding a methyl group at C24 of the sterol precursor cycloartenol. SMT2 and SMT3 act at later steps and were shown to be functionally redundant as C-24 sterol methyltransferases at the branching in sterol synthesis that either leads to sitosterol or campesterol (21). The total sterol composition in smt1 mutants was shown to be different from wild type, with the major phytosterols like sitosterol, stigmasterol, and brassicasterol being strongly depleted. In contrast, other sterol species remained unaltered and some even increased (20, 21). So far, it remains unclear how the altered sterol-composition of the smt1 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 mβcd treatment and on endogenous alterations in sterol composition in smt1 to improve understanding of sterol–protein interactions. |
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