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Property-controlling Enzymes at the Membrane Interface
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Åke Wieslander)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Monotopic proteins represent a specialized group of membrane proteins in that they are engaged in biochemical events taking place at the membrane interface. In particular, the monotopic lipid-synthesizing enzymes are able to synthesize amphiphilic lipid products by catalyzing two biochemically distinct molecules (substrates) at the membrane interface. Thus, from an evolutionary point of view, anchoring into the membrane interface enables monotopic enzymes to confer sensitivity to a changing environment by regulating their activities in the lipid biosynthetic pathways in order to maintain a certain membrane homeostasis. We are focused on a plant lipid-synthesizing enzyme DGD2 involved in phosphate shortage stress, and analyzed the potentially important lipid anchoring segments of it, by a set of biochemical and biophysical approaches. A mechanism was proposed to explain how DGD2 adjusts its activity to maintain a proper membrane. In addition, a multivariate-based bioinformatics approach was used to predict the lipid-binding segments for GT-B fold monotopic enzymes. In contrast, a soluble protein Myr1 from yeast, implicated in vesicular traffic, was also proposed to be a membrane stress sensor as it is able to exert different binding properties to stressed membranes, which is probably due to the presence of strongly plus-charged clusters in the protein. Moreover, a bacterial monotopic enzyme MGS was found to be able to induce massive amounts of intracellular vesicles in Escherichia coli cells. The mechanisms involve several steps: binding, bilayer lateral expansion, stimulation of lipid synthesis, and membrane bending. Proteolytic and mutant studies indicate that plus-charged residues and the scaffold-like structure of MGS are crucial for the vesiculation process. Hence, a number of features are involved governing the behaviour of monotopic membrane proteins at the lipid bilayer interface.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2011. , 80 p.
Keyword [en]
monotopic membrane protein, lipid-protein interaction, membrane curvature, glycosyltransferase, Rossmann fold
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-61988ISBN: 978-91-7447-330-8 (print)OAI: oai:DiVA.org:su-61988DiVA: diva2:439223
Public defence
2011-10-21, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.Available from: 2011-09-29 Created: 2011-09-06 Last updated: 2011-11-18Bibliographically approved
List of papers
1. Tryptophan residues promote membrane association for a plant lipid glycosyltransferase involved in phosphate stress
Open this publication in new window or tab >>Tryptophan residues promote membrane association for a plant lipid glycosyltransferase involved in phosphate stress
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 8, 6669-6684 p.Article in journal (Refereed) Published
Abstract [en]

Chloroplast membranes contain a substantial excess of the nonbilayer-prone monogalactosyldiacylglycerol (GalDAG) over the biosynthetically consecutive, bilayer-forming digalactosyldiacylglycerol (GalGalDAG), yielding a high membrane curvature stress. During phosphate shortage, plants replace phospholipids with GalGalDAG to rescue phosphate while maintaining membrane homeostasis. Here we investigate how the activity of the corresponding glycosyltransferase (GT) in Arabidopsis thaliana (atDGD2) depends on local bilayer properties by analyzing structural and activity features of recombinant protein. Fold recognition and sequence analyses revealed a two-domain GT-B monotopic structure, present in other plant and bacterial glycolipid GTs, such as the major chloroplast GalGalDAG GT atDGD1. Modeling led to the identification of catalytically important residues in the active site of atDGD2 by site-directed mutagenesis. The DGD synthases share unique bilayer interface segments containing conserved tryptophan residues that are crucial for activity and for membrane association. More detailed localization studies and liposome binding analyses indicate differentiated anchor and substrate-binding functions for these separated enzyme interface regions. Anionic phospholipids, but not curvature-increasing nonbilayer lipids, strongly stimulate enzyme activity. From our studies, we propose a model for bilayer "control" of enzyme activity, where two tryptophan segments act as interface anchor points to keep the substrate region close to the membrane surface. Binding of the acceptor substrate is achieved by interaction of positive charges in a surface cluster of lysines, arginines, and histidines with the surrounding anionic phospholipids. The diminishing phospholipid fraction during phosphate shortage stress will then set the new GalGalDAG/phospholipid balance by decreasing stimulation of atDGD2.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62029 (URN)10.1074/jbc.M110.138495 (DOI)000287476400075 ()
Available from: 2011-09-07 Created: 2011-09-07 Last updated: 2017-12-08Bibliographically approved
2. Lipid Interacting Regions in Phosphate Stress Glycosyltransferase atDGD2 from Arabidopsis thaliana
Open this publication in new window or tab >>Lipid Interacting Regions in Phosphate Stress Glycosyltransferase atDGD2 from Arabidopsis thaliana
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2011 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 21, 4451-4466 p.Article in journal (Refereed) Published
Abstract [en]

Membrane lipid glycosyltransferases (GTs) in plants are enzymes that regulate the levels of the non-bilayer prone monogalactosyldiacylglycerol (GalDAG) and the bilayer-forming digalactosyldiacylglycerol (GalGalDAG). The relative amounts of these lipids affect membrane properties such as curvature and lateral stress. During phosphate shortage, phosphate is rescued by replacing phospholipids with GalGalDAG. The glycolsyltransferase enzyme in Arabidopsis thaliana responsible for this, atDGD2, senses the bilayer properties and interacts with the membrane in a monotopic manner. To understand the parameters that govern this interaction, we have identified several possible lipid-interacting sites in the protein and studied these by biophysical techniques. We have developed a multivariate discrimination algorithm that correctly predicts the regions in the protein that interact with lipids, and the interactions were confirmed by a variety of biophysical techniques. We show by bioinformatic methods and circular dichroism (CD), fluorescence, and NMR spectroscopic techniques that two regions are prone to interact with lipids in a surface-charge dependent way. Both of these regions contain Trp residues, but here charge appears to be the dominating feature governing the interaction. The sequence corresponding to residues 227–245 in the protein is seen to be able to adapt its structure according to the surface-charge density of a bilayer. All results indicate that this region interacts specifically with lipid molecules and that a second region in the protein, corresponding to residues 130–148, also interacts with the bilayer. On the basis of this, and sequence charge features in the immediate environment of S227–245, a response model for the interaction of atDGD2 with the membrane bilayer interface is proposed.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62012 (URN)10.1021/bi200162f (DOI)000290837400008 ()
Funder
Swedish Research Council, 621-2011-5964
Available from: 2011-09-07 Created: 2011-09-07 Last updated: 2017-12-08
3. Basic clusters and amphipathic helices contribute to interactions of Myr1/Syh1 with membrane phospholipids
Open this publication in new window or tab >>Basic clusters and amphipathic helices contribute to interactions of Myr1/Syh1 with membrane phospholipids
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The ability to associate transiently with membrane bilayers is an important property of many protein regulators of membrane trafficking, lipid transfer proteins, or signaling modules. Membrane association is also a property of Myr1/Syh1, a soluble GYF domain protein from Saccharomyces cerevisiae, previously reported to rescue the temperature sensitive growth of ypt6 and ric1 null strains. Here, we further demonstrate that MYR1 also rescued the vacuole fragmentation phenotype of the ypt6 and ric1 mutants. The mechanism behind these genetic interactions is likely linked to the capacity of the Myr1/Syh1 protein to associate with phospholipid membranes. In order to elucidate further the nature of the interactions with vesicular traffic, we studied protein-protein and protein-phospholipid association of isolated domains from Myr1/Syh1. Using a two-hybrid assay, we confirmed the capacity of Myr1/Syh1 to self-associate in vivo. We measured in vitro the affinity of recombinant Myr1/Syh1 domains fused to GFP for liposomes reconstituted from synthetic and natural yeast lipids by sedimentation techniques. The herewith established affinities of Myr1/Syh1 to specific lipids, combined with evidence for its interactions with membrane traffic and protein synthesis, provide support for a possible function of Myr1/Syh1 as a regulator sensing membrane composition along the vesicular pathways.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62030 (URN)
Available from: 2011-09-07 Created: 2011-09-07 Last updated: 2011-09-08Bibliographically approved
4. Massive formation of intracellular membrane vesicles in Escherichia coli by a monotopic membrane-bound lipid glycosyltransferase
Open this publication in new window or tab >>Massive formation of intracellular membrane vesicles in Escherichia coli by a monotopic membrane-bound lipid glycosyltransferase
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2009 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 49, 33904-33914 p.Article in journal (Refereed) Published
Abstract [en]

The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of > or = 60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter approximately 100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-34581 (URN)10.1074/jbc.M109.021618 (DOI)000272165200023 ()19767390 (PubMedID)
Available from: 2010-01-11 Created: 2010-01-11 Last updated: 2017-12-12Bibliographically approved
5. Modulation of Escherichia coli Cell Membrane by a Monotopic Lipid Glycosyltransferase - an Exploration of Potential Mechanisms
Open this publication in new window or tab >>Modulation of Escherichia coli Cell Membrane by a Monotopic Lipid Glycosyltransferase - an Exploration of Potential Mechanisms
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Intracellular vesicles are abundant in eukaryotic cells but are rare in Gram-negative bacterium Escherichia coli. Strongly overexpression of a monotopic glycolipid-synthesizing enzyme could induce massive formation of “foreign” vesicles in the cytoplasm. Here we investigate how this membrane-associated enzyme is able to bend and deform the plasma membrane. Limited proteolysis combined with ESI-MS suggested interface binding is mediated through both its two Rossmann fold topological domains. Detailed subcellular localization and liposome binding assay indicates different interface anchoring regions in the protein, and anionic lipid seems to influence the binding properties of the anchoring segments. Genetic engineering of a known membrane-bound segment to explore its vesiculation potentials led to the identification of important catalytic residues (regions). Flow cytometry and infrared spectroscopy were also performed on bacterial cells to get more insight into the cellular morphology and internal complexity. The linking region between two domains was demonstrated to be crucial for both catalytic function and vesiculation capacity of the enzyme. Based on our findings, we propose, that scaffold-like structural feature of this enzyme is most likey one of key elements contributing to vesiculation.        

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62031 (URN)
Available from: 2011-09-07 Created: 2011-09-07 Last updated: 2011-09-08Bibliographically approved

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