Validity and applicability of membrane model systems for studying interactions of peripheral membrane proteins with lipids

Czogalla, Aleksander; Grzybek, Michał; Jones, Walis; Coskun, Ünal

doi:10.1016/j.bbalip.2013.12.012

Cited by 62 publications

(58 citation statements)

References 150 publications

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“…Despite the fact that analysis of the PI-binding proteome by quantitative mass spectrometry identified MTM1 as a PI(4)P-specific binder (Jungmichel, Sylvestersen et al 2014), liposome flotation assays at least in our hands failed to reveal selective binding to PI(4)P. Instead, MTM1 preferentially bound to its substrate PI (3) shown to be essential for membrane association of PI4K2α (Lu, Sun et al 2012). It could further impact the presentation of lipid head groups (Czogalla, Grzybek et al 2014), might define PI4K2α-enriched endosomal subdomains and thus, modulate MTM1-lipid binding.…”

Section: Membrane Recruitment Of Mtm1 Is Regulated By Lipid and Protementioning

confidence: 54%

A phosphoinositide conversion mechanism for exit from endosomes

Ketel¹,

Krauß²,

Nicot³

et al. 2016

Nature

161

177

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I owe special thanks to Marnix Wieffer and Michael Krauß, who spent a lot of time helping me to get started in the lab, to overcome numerous experimental problem, I was not sure how to deal with, and frequently joined in discussions about my project with excellent advice. I am really grateful for your experimental support, Michael, and the time you invested in the project during our paper revision.Further, I would like to thank Jocelyn Laporte and his group, especially Anne-Sophie Nicot, at the IGBMC in Strasbourg for their support and a very fruitful collaboration, and Carsten Schultz and his group at the EMBL in Heidelberg for their support and constant supply with PIP/AMs. I owe special thanks to Dmytro Puchkov for the ultrastructural analysis and Eberhard Krause for mass spectrometry done within this project. I also need to acknowledge Markus Wenk and Federico Torta at the Singapore Lipidomics Incubator for joining the project at a very late stage, when we urgently needed help from lipidomics specialists. It was a pity that experiments did not work out as planned.I would further like to express my gratitude to two very skilled technicians in the AG Haucke lab: Silke Zillmann and Delia Löwe. Without your help it would have been impossible to achieve so much within the limited amount of time I had during the paper revision. by VPS34-IN1 treatment................................................... 52 2.3.11 Fluorescence microscopy................................................................................. 53 2.3.12 Flow cytometry............................................................................................ III SummaryPhosphoinositides (PIs) are a minor class of short-lived phospholipids that serve as crucial signposts of membrane identity. Thereby, PIs full fill important functions in cell signaling and membrane transport. PI 4-phosphates such as phosphatitylinositol-4-phosphate (PI(4)P) and phosphatitylinositol-4,5-bisphosphate (PI(4,5)P 2 ) are enriched at the plasma membrane (PM), on secretory organelles and lysosomes, while PI 3-phosphates, i.e.phosphatitylinositol-3-phosphate (PI(3)P), are a hallmark of the endosomal system.Directional transport between these compartments, thus, requires regulated PI conversion.However, PI conversion in exit from PI(3)P-enriched endosomes en route to the PI(4)P-and PI(4,5)P 2 -positive PM in endosomal recycling remained unknown.Here, we report that cargo exit from endosomes requires removal of PI(3)P by the PI(3)P 3-phosphatase myotubularin 1 (MTM1), and concomitant PI(4)P synthesis by PI 4-kinase type II α (PI4K2α). Loss of MTM1 causes endosomal accumulation of PI(3)P and PI(3)P effector proteins, i.e. sorting nexins, Kif16b-mediated outward traffic of PI(3)P containing endosomes and sub-plasmalemmal accumulation of exocytosis-deficient endosomes. As PI4K2α associates with MTM1 and thereby facilitates membrane recruitment of MTM1, these phenotypic changes are mimicked by loss of PI4K2α. The conversion of PI(3)P-to-PI(4)P is paralleled by a switch i...

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Section: Membrane Recruitment Of Mtm1 Is Regulated By Lipid and Protementioning

confidence: 54%

A phosphoinositide conversion mechanism for exit from endosomes

Ketel¹,

Krauß²,

Nicot³

et al. 2016

Nature

161

177

View full text Add to dashboard Cite

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“…As a consequence, the physicochemical properties of their membranes vary significantly throughout the cell (52), exemplified by the plasma membrane being highly enriched in cholesterol (∼35-40 mol%) and sphingolipids, both critically regulating membrane fluidity and thickness (53). Upon cholesterol depletion, the EGFR kinase domain is activated in a ligand-independent manner (24,54).…”

Section: Discussionmentioning

confidence: 99%

N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

Kaszuba

Grzybek

Orłowski

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

143

156

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The epidermal growth factor receptor (EGFR) regulates several critical cellular processes and is an important target for cancer therapy. In lieu of a crystallographic structure of the complete receptor, atomistic molecular dynamics (MD) simulations have recently shown that they can excel in studies of the full-length receptor. Here we present atomistic MD simulations of the monomeric N-glycosylated human EGFR in biomimetic lipid bilayers that are, in parallel, also used for the reconstitution of full-length receptors. This combination enabled us to experimentally validate our simulations, using ligand binding assays and antibodies to monitor the conformational properties of the receptor reconstituted into membranes. We find that N-glycosylation is a critical determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. In the absence of a structure for full-length, posttranslationally modified membrane receptors, our approach offers new means to structurally define and experimentally validate functional properties of cell surface receptors in biomimetic membrane environments.R eceptor tyrosine kinases (RTKs) are cell surface receptors that receive and transduce signals mediating a variety of critical cellular processes, including cell growth, migration, proliferation, differentiation, and apoptosis. Among the many RTKs, the most studied is the epidermal growth factor receptor (EGFR), not least because of its involvement in the development and progression of epidermoid cancers and its resulting importance as a target for antineoplastic therapies.Structurally, the EGFR consists of the ectodomain (ECD) (further subdivided into four subdomains, DI-IV), the transmembrane domain (TMD), and the intracellular tyrosine kinase domain (TKD). Ligand binding induces conformational transitions of the ECD that stabilize receptor dimerization, culminating in the activation of the intracellular TKD and subsequent propagation of the activation signal (1). To prevent receptor activation and signaling in the absence of ligand, the structurally tethered ECD of monomeric EGFR blocks the intrinsic capacity of the TMD and the intracellular TKD to dimerize (2). Ligand binding is believed to release the self-inhibitory tether and facilitate receptor oligomerization and activation (3-6). A detailed understanding of the structural regulation of the intact full-length receptors in their native membranes promises to reveal the molecular basis for receptor regulation (7); however, the methodological limitations associated with crystallizing transmembrane proteins, together with the high flexibility of the full-length receptor, have prevented high-resolution crystallographic analysis.To fill this gap, extensive atom-scale molecular dynamics (MD) simulations were recently performed to elucidate the structural dynamics of the EGFR in a two-component lipid bilayer (8). These studies suggest a large interfacial contact area between the membrane and the ecto-and intracellular domains of the unli...

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“…However, a shortcoming of these methods is the non-native environment of the GLs, which could influence the nature of protein-GL interactions. An alternative approach is to incorporate the GLs in a lipid monolayer or bilayer, such that the protein-GL interactions can be studied in a more native-like environment [10]. For such studies, a variety of different model membranes have been used to solubilize the GLs, including supported lipid bilayers, liposomes, micelles, bicelles, nanodiscs, and picodiscs [11][12][13][14], and the protein-GL interactions probed using diverse analytical techniques (e.g., fluorescence, nuclear magnetic resonance (NMR), and SPR spectroscopy) [15][16][17][18].between water-soluble lectins and GLs, solubilized using nanodiscs (NDs), have been detected using the catch-andrelease (CaR)-ESI-MS assay [19,20].…”

Section: Introductionmentioning

confidence: 99%

Detecting Protein–Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS

Han

Kitova

Klassen

2016

J. Am. Soc. Mass Spectrom.

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Abstract. This study reports on the use of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay, combined with glycomicelles, as a method for detecting specific interactions between water-soluble proteins and glycolipids (GLs) in aqueous solution. The B subunit homopentamers of cholera toxin (CTB 5 ) and Shiga toxin type 1 B (Stx1B 5 ) and the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2 served as model systems for this study. The CTB 5 exhibits broad specificity for gangliosides and binds to GM1, GM2, GM3, GD1a, GD1b, and GT1b; Stx1B 5 does not recognize gangliosides. The CaR-ESI-MS assay was used to analyze solutions of CTB 5 or Stx1B 5 and individual gangliosides (GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2) or mixtures thereof. The high affinity interaction of CTB 5 with GM1 was successfully detected. However, the apparent affinity, as determined from the mass spectra, is significantly lower than that of the corresponding pentasaccharide or when GM1 is presented in model membranes such as nanodiscs. Interactions between CTB 5 and the low affinity gangliosides GD1a, GD1b, and GT1b, as well as GD2, which served as a negative control, were detected; no binding of CTB 5 to GM2 or GM3 was observed. The CaR-ESI-MS results obtained for Stx1B 5 reveal that nonspecific protein-ganglioside binding can occur during the ESI process, although the extent of binding varies between gangliosides. Consequently, interactions detected for CTB 5 with GD1a, GD1b, and GT1b are likely nonspecific in origin. Taken together, these results reveal that the CaR-ESI-MS/glycomicelle approach for detecting protein-GL interactions is prone to false positives and false negatives and must be used with caution.

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Validity and applicability of membrane model systems for studying interactions of peripheral membrane proteins with lipids

Cited by 62 publications

References 150 publications

A phosphoinositide conversion mechanism for exit from endosomes

A phosphoinositide conversion mechanism for exit from endosomes

N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

Detecting Protein–Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS

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