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Modulation of Escherichia coli Cell Membrane by a Monotopic Lipid Glycosyltransferase - an Exploration of Potential Mechanisms
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Åke Wieslander)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
(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: urn:nbn:se:su:diva-62031OAI: oai:DiVA.org:su-62031DiVA: diva2:439466
Available from: 2011-09-07 Created: 2011-09-07 Last updated: 2011-09-08Bibliographically approved
In thesis
1. Property-controlling Enzymes at the Membrane Interface
Open this publication in new window or tab >>Property-controlling Enzymes at the Membrane Interface
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
monotopic membrane protein, lipid-protein interaction, membrane curvature, glycosyltransferase, Rossmann fold
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-61988 (URN)978-91-7447-330-8 (ISBN)
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

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