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1.
Angew Chem Int Ed Engl ; 57(40): 13339-13343, 2018 10 01.
Article in English | MEDLINE | ID: mdl-30048020

ABSTRACT

Lipid messengers exert their function on short time scales at distinct subcellular locations, yet most experimental approaches for perturbing their levels trigger cell-wide concentration changes. Herein, we report on a coumarin-based photocaging group that can be modified with organelle-targeting moieties by click chemistry and thus enables photorelease of lipid messengers in distinct organelles. We show that caged arachidonic acid and sphingosine derivatives can be selectively delivered to mitochondria, the ER, lysosomes, and the plasma membrane. By comparing the cellular calcium transients induced by localized uncaging of arachidonic acid and sphingosine, we show that the precise intracellular localization of the released second messenger is crucial for the signaling outcome. Ultimately, we anticipate that this new class of caged compounds will greatly facilitate the study of cellular processes on the organelle level.


Subject(s)
Arachidonic Acid/chemistry , Click Chemistry , Coumarins/chemistry , Organelles/metabolism , Sphingosine/analogs & derivatives , Arachidonic Acid/metabolism , Calcium/metabolism , Coumarins/metabolism , Endoplasmic Reticulum/metabolism , HeLa Cells , Humans , Lysosomes/metabolism , Microscopy, Fluorescence , Mitochondria/metabolism , Sphingosine/metabolism , Time-Lapse Imaging , Ultraviolet Rays
2.
Proc Natl Acad Sci U S A ; 112(10): 3092-7, 2015 Mar 10.
Article in English | MEDLINE | ID: mdl-25733861

ABSTRACT

The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichous-flagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.


Subject(s)
Bacterial Proteins/physiology , Flagella/physiology , Bacterial Proteins/metabolism , Dimerization , Flagella/enzymology
3.
Analyst ; 139(8): 1987-92, 2014 Apr 21.
Article in English | MEDLINE | ID: mdl-24599267

ABSTRACT

Small molecule sensing is of great importance in pharmaceutical research. While there exist well established screening methods such as EMSA (electrophoretic motility shift assay) or biointeraction chromatography to report on successful binding interactions, there are only a few techniques that allow studying and quantifying the interaction of low molecular weight analytes with a binding partner directly. We report on a binding assay for small molecules based on the reflectivity change of a porous transparent film upon immobilisation of an absorbing substance on the pore walls. The porous matrix acts as a thin optical transparent film to produce interference fringes and accumulates molecules at the inner wall to amplify the sensor response. The benefits and limits of the assay are demonstrated by investigating the binding of biotin labelled with an atto dye to avidin physisorbed within an anodic aluminium oxide membrane.


Subject(s)
Proteins/chemistry , Electrophoretic Mobility Shift Assay , Molecular Weight
4.
Anal Chem ; 86(3): 1366-71, 2014 Feb 04.
Article in English | MEDLINE | ID: mdl-24377291

ABSTRACT

The passage of solutes across a lipid membrane plays a central role in many cellular processes. However, the investigation of transport processes remains a serious challenge in pharmaceutical research, particularly the transport of uncharged cargo. While translocation reactions of ions across cell membranes is commonly measured with the patch-clamp, an equally powerful screening method for the transport of uncharged compounds is still lacking. A combined setup for reflectometric interference spectroscopy (RIfS) and fluorescence microscopy measurements is presented that allows one to investigate the passive exchange of uncharged compounds across a free-standing membrane. Pore-spanning lipid membranes were prepared by spreading giant 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles on porous anodic aluminum oxide (AAO) membranes, creating sealed attoliter-sized compartments. The time-resolved leakage of different dye molecules (pyranine and crystal violet) as well as avidin through melittin induced membrane pores and defects was investigated.


Subject(s)
Cell Membrane/metabolism , Microscopy, Fluorescence , Spectrum Analysis , Aluminum Oxide/chemistry , Cell Membrane/chemistry , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Melitten/metabolism , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Protein Transport , Unilamellar Liposomes/chemistry , Unilamellar Liposomes/metabolism
5.
PLoS One ; 9(1): e85033, 2014.
Article in English | MEDLINE | ID: mdl-24392163

ABSTRACT

Tail-Anchored (TA) proteins are inserted into the endoplasmic reticulum (ER) membrane of yeast cells via the posttranslational Guided Entry of Tail-Anchored protein (GET) pathway. The key component of this targeting machinery is the ATPase Get3 that docks to the ER membrane by interacting with a receptor complex formed by the proteins Get1 and Get2. A conserved pathway is present in higher eukaryotes and is mediated by TRC40, homolog of Get3, and the recently identified membrane receptors WRB and CAML. Here, we used yeast lacking the GET1 and GET2 genes and substituted them with WRB and CAML. This rescued the growth phenotypes of the GET receptor mutant. We demonstrate that WRB and CAML efficiently recruit Get3 to the ER membrane and promote the targeting of the TA proteins in vivo. Our results show that the membrane spanning segments of CAML are essential to create a functional receptor with WRB and to ensure TA protein membrane insertion. Finally, we determined the binding parameters of TRC40 to the WRB/CAML receptor. We conclude that together, WRB and CAML are not only necessary but also sufficient to create a functional membrane receptor complex for TRC40. The yeast complementation assay can be used to further dissect the structure-function relationship of the WRB/CAML heteromultimer in the absence of endogenous receptor proteins.


Subject(s)
Endoplasmic Reticulum/metabolism , Intracellular Membranes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Phenotype , Protein Interaction Domains and Motifs , Protein Transport , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
6.
Autophagy ; 9(5): 770-7, 2013 May.
Article in English | MEDLINE | ID: mdl-23445924

ABSTRACT

We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P 2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.


Subject(s)
Biochemistry/methods , Carrier Proteins/metabolism , Liposomes/metabolism , Phosphatidylinositol Phosphates/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Calorimetry , Fractionation, Field Flow , Light , Protein Binding , Scattering, Radiation
7.
Autophagy ; 9(1): 106-7, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23069643

ABSTRACT

PROPPINs are a family of PtdIns3P and PtdIns(3,5)P 2-binding proteins. The crystal structure now unravels the presence of two distinct phosphoinositide-binding sites at the circumference of the seven bladed ß-propeller. Mutagenesis analysis of the binding sites shows that both are required for normal membrane association and autophagic activities. We identified a set of evolutionarily conserved basic and polar residues within both binding pockets, which are crucial for phosphoinositide binding. We expect that membrane association of PROPPINs is further stabilized by membrane insertions and interactions with other proteins.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Phosphatidylinositols/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Binding Sites , Carrier Proteins/genetics , Crystallography, X-Ray , Kluyveromyces/metabolism , Models, Molecular , Mutagenesis , Phosphatidylinositol Phosphates/genetics , Phosphatidylinositol Phosphates/metabolism , Phosphatidylinositols/genetics , Protein Conformation , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
8.
Proc Natl Acad Sci U S A ; 109(30): E2042-9, 2012 Jul 24.
Article in English | MEDLINE | ID: mdl-22753491

ABSTRACT

ß-propellers that bind polyphosphoinositides (PROPPINs), a eukaryotic WD-40 motif-containing protein family, bind via their predicted ß-propeller fold the polyphosphoinositides PtdIns3P and PtdIns(3,5)P(2) using a conserved FRRG motif. PROPPINs play a key role in macroautophagy in addition to other functions. We present the 3.0-Å crystal structure of Kluyveromyces lactis Hsv2, which shares significant sequence homologies with its three Saccharomyces cerevisiae homologs Atg18, Atg21, and Hsv2. It adopts a seven-bladed ß-propeller fold with a rare nonvelcro propeller closure. Remarkably, in the crystal structure, the two arginines of the FRRG motif are part of two distinct basic pockets formed by a set of highly conserved residues. In comprehensive in vivo and in vitro studies of ScAtg18 and ScHsv2, we define within the two pockets a set of conserved residues essential for normal membrane association, phosphoinositide binding, and biological activities. Our experiments show that PROPPINs contain two individual phosphoinositide binding sites. Based on docking studies, we propose a model for phosphoinositide binding of PROPPINs.


Subject(s)
Kluyveromyces/chemistry , Membrane Proteins/chemistry , Models, Molecular , Protein Conformation , Saccharomyces cerevisiae Proteins/chemistry , Amino Acid Motifs/genetics , Autophagy-Related Proteins , Binding Sites/genetics , Cloning, Molecular , Conserved Sequence/genetics , Crystallography, X-Ray , Membrane Proteins/genetics , Molecular Dynamics Simulation , Mutagenesis , Phosphatidylinositols/metabolism , Protein Binding , Saccharomyces cerevisiae Proteins/genetics
9.
J Am Chem Soc ; 134(7): 3326-9, 2012 Feb 22.
Article in English | MEDLINE | ID: mdl-22296574

ABSTRACT

The multivalent carbohydrate-carbohydrate interaction between membrane-anchored epitopes derived from the marine sponge Microciona prolifera has been explored by colloidal probe microscopy. An in situ coupling of sulfated and non-sulfated disaccharides to membrane-coated surfaces was employed to mimic native cell-cell contacts.The dynamic strength of the homomeric self-association was measured as a function of calcium ions and loading rate. A deterministic model was used to estimate the basic energy landscape and number of participating bonds in the contact zone.


Subject(s)
Disaccharides/chemistry , Lipid Bilayers/chemistry , Porifera/chemistry , Animals , Cell Adhesion , Disaccharides/isolation & purification , Microscopy, Scanning Probe
10.
J Am Chem Soc ; 131(20): 7031-9, 2009 May 27.
Article in English | MEDLINE | ID: mdl-19453196

ABSTRACT

The mechanical behavior of lipid bilayers spanning the pores of highly ordered porous silicon substrates was scrutinized by local indentation experiments as a function of surface functionalization, lipid composition, solvent content, indentation velocity, and pore radius. Solvent-containing nano black lipid membranes (nano-BLMs) as well as solvent-free pore-spanning bilayers were imaged by fluorescence and atomic force microscopy prior to force curve acquisition, which allows distinguishing between membrane-covered and uncovered pores. Force indentation curves on pore-spanning bilayers attached to functionalized hydrophobic porous silicon substrates reveal a predominately linear response that is mainly attributed to prestress in the membranes. This is in agreement with the observation that indentation leads to membrane lysis well below 5% area dilatation. However, membrane bending and lateral tension dominate over prestress and stretching if solvent-free supported membranes obtained from spreading giant liposomes on hydrophilic porous silicon are indented. An elastic regime diagram is presented that readily allows determining the dominant contribution to the mechanical response upon indentation as a function of load and pore radius.


Subject(s)
Lipid Bilayers/chemistry , Gold/chemistry , Hydrophobic and Hydrophilic Interactions , Membranes/chemistry , Microscopy, Atomic Force , Microscopy, Fluorescence , Models, Chemical , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/chemistry , Silicon/chemistry , Stress, Mechanical
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