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Exploring the Interplay of Lipids and Membrane Proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The interplay between lipids and membrane proteins is known to affect membrane protein topology and thus have significant effect (control) on their functions. In this PhD thesis, the influence of lipids on the membrane protein function was studied using three different membrane protein models.

A monotopic membrane protein, monoglucosyldiacylglyecerol synthase (MGS) from Acholeplasma laidlawii is known to induce intracellular vesicles when expressed in Escherichia coli. The mechanism leading to this unusual phenomenon was investigated by various biochemical and biophysical techniques. The results indicated a doubling of lipid synthesis in the cell, which was triggered by the selective binding of MGS to anionic lipids. Multivariate data analysis revealed a good correlation with MGS production. Furthermore, preferential anionic lipid sequestering by MGS was shown to induce a different fatty acid modeling of E. coli membranes. The roles of specific lipid binding and the probable mechanism leading to intracellular vesicle formation were also investigated.

As a second model, a MGS homolog from Synechocystis sp. PCC6803 was selected. MgdA is an integral membrane protein with multiple transmembrane helices and a unique membrane topology. The influence of different type of lipids on MgdA activity was tested with different membrane fractions of Synechocystis. Results indicated a very distinct profile compared to Acholeplasma laidlawii MGS. SQDG, an anionic lipid was found to be the species of the membrane that increased the MgdA activity 7-fold whereas two other lipids (PG and PE) had only minor effects on MgdA. Additionally, a working model of MgdA for the biosynthesis and flow of sugar lipids between Synechocystis membranes was proposed.

The last model system was another integral membrane protein with a distinct structure but also a different function. The envelope stress sensor, CpxA and its interaction with E. coli membranes were studied. CpxA autophosphorylation activity was found to be positively regulated by phosphatidylethanolamine and negatively by anionic lipids. In contrast, phosphorylation of CpxR by CpxA revealed to be increased with PG but inhibited by CL. Non-bilayer lipids had a negative impact on CpxA phosphotransfer activity.

Taken together, these studies provide a better understanding of the significance of the interplay of lipids and model membrane proteins discussed here.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2014. , 69 p.
Keyword [en]
Membrane lipids, membrane proteins, anionic lipids, membrane remodeling, intracellular vesicles, model membrane systems, glycosyltransferase, Escherichia coli, lipid composition, fatty acid modification, membrane curvature, bacterial homeoviscous adaptation
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-102675ISBN: 978-91-7447-882-2 (print)OAI: oai:DiVA.org:su-102675DiVA: diva2:712361
Public defence
2014-05-16, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Foundation for Strategic Research Swedish Research Council
Available from: 2014-04-24 Created: 2014-04-14 Last updated: 2015-03-31Bibliographically approved
List of papers
1. Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli
Open this publication in new window or tab >>Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli
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2013 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 33, 5533-5544 p.Article in journal (Refereed) Published
Abstract [en]

Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by P-31 NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a feed-forward stimulation of lipid synthesis in E. coil. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry; Biophysics
Identifiers
urn:nbn:se:su:diva-93756 (URN)10.1021/bi400616n (DOI)000323471900005 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

AuthorCount:9;

Available from: 2013-09-20 Created: 2013-09-16 Last updated: 2017-12-06Bibliographically approved
2. Heterologous overexpression of a monotopic glucosyltransferase (MGS) induces fatty acid remodeling in Escherichia coli membranes:  
Open this publication in new window or tab >>Heterologous overexpression of a monotopic glucosyltransferase (MGS) induces fatty acid remodeling in Escherichia coli membranes:  
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2014 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 7, 1862-1870 p.Article in journal (Refereed) Published
Abstract [en]

The membrane protein monoglucosyldiacylglycerol synthase (MGS) from Acholeplasma laidlawii is responsible for the creation of intracellular membranes when overexpressed in Escherichia coli (E. coli). The present study investigates time dependent changes in composition and properties of E. coli membranes during 22 h of MGS induction. The lipid/protein ratio increased by 38% in MGS-expressing cells compared to control cells. Time-dependent screening of lipids during this period indicated differences in fatty acid modeling. (1) Unsaturation levels remained constant for MGS cells (~ 62%) but significantly decreased in control cells (from 61% to 36%). (2) Cyclopropanated fatty acid content was lower in MGS producing cells while control cells had an increased cyclopropanation activity. Among all lipids, phosphatidylethanolamine (PE) was detected to be the most affected species in terms of cyclopropanation. Higher levels of unsaturation, lowered cyclopropanation levels and decreased transcription of the gene for cyclopropane fatty acid synthase (CFA) all indicate the tendency of the MGS protein to force E. coli membranes to alter its usual fatty acid composition.

Keyword
Lipid–protein interactions, fatty acid modeling, cyclopropanation, membrane fluidity, monotopic membrane proteins, intracellular membranes
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-102674 (URN)10.1016/j.bbamem.2014.04.001 (DOI)000336695300020 ()
Funder
Swedish Foundation for Strategic Research Swedish Research Council, 621-2006-4818Knut and Alice Wallenberg FoundationCarl Tryggers foundation Swedish Research Council, 2008-19122-62107-3Swedish Research Council, 621-2011-3524
Available from: 2014-04-14 Created: 2014-04-14 Last updated: 2017-12-05Bibliographically approved
3. Subcellular Localization of Monoglucosyldiacylglycerol Synthase in Synechocystis sp PCC6803 and Its Unique Regulation by Lipid Environment
Open this publication in new window or tab >>Subcellular Localization of Monoglucosyldiacylglycerol Synthase in Synechocystis sp PCC6803 and Its Unique Regulation by Lipid Environment
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 2, e88153- p.Article in journal (Refereed) Published
Abstract [en]

Synthesis of monogalactosyldiacylglycerol (GalDAG) and digalactosyldiacylglycerol (GalGalDAG), the major membrane lipids in cyanobacteria, begins with production of the intermediate precursor monoglucosyldiacylglycerol (GlcDAG), by monoglucosyldiacylglycerol synthase (MGS). In Synechocystis sp. PCC6803 (Synechocystis) this activity is catalyzed by an integral membrane protein, Sll1377 or MgdA. In silico sequence analysis revealed that cyanobacterial homologues of MgdA are highly conserved and comprise a distinct group of lipid glycosyltransferases. Global regulation of lipid synthesis in Synechocystis and, more specifically, the influence of the lipid environment on MgdA activity have not yet been fully elucidated. Therefore, we purified membrane subfractions from this organism and assayed MGS activity in vitro, with and without different lipids and other potential effectors. Sulfoquinovosyldiacylglycerol (SQDAG) potently stimulates MgdA activity, in contrast to other enzymes of a similar nature, which are activated by phosphatidylglycerol instead. Moreover, the final products of galactolipid synthesis, GalDAG and GalGalDAG, inhibited this activity. Western blotting revealed the presence of MgdA both in plasma and thylakoid membranes, with a high specific level of the MgdA protein in the plasma membrane but highest MGS activity in the thylakoid membrane. This discrepancy in the subcellular localization of enzyme activity and protein may indicate the presence of either an unknown regulator and/or an as yet unidentified MGS-type enzyme. Furthermore, the stimulation of MgdA activity by SQDAG observed here provides a new insight into regulation of the biogenesis of both sulfolipids and galactolipids in cyanobacteria.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-101739 (URN)10.1371/journal.pone.0088153 (DOI)000330830700049 ()
Funder
Swedish Foundation for Strategic Research
Note

AuthorCount:5;

Funding agencies:

NTU (Singapore) M4080306;

Available from: 2014-03-17 Created: 2014-03-14 Last updated: 2017-12-05Bibliographically approved
4. The Escherichia coli envelope stress sensor CpxA can sense changes in lipid bilayer properties
Open this publication in new window or tab >>The Escherichia coli envelope stress sensor CpxA can sense changes in lipid bilayer properties
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Membrane proteins are evolutionarily integrated with their lipid bilayer environment, but it is only marginally understood how they sense and adapt to changes in lipid composition. Here we use an in vitro reconstitution system to demonstrate that the activity of envelope stress sensor CpxA of Escherichia coli is modulated by lipid bilayer properties. By changing the lipid composition of the vesicles, and using lipid-engineered E. coli strains we show that CpxA activity is regulated by means of different properties of the bilayer: (i) anionic surface charge (ii) membrane curvature (iii) thus lateral bilayer stress but also (iv) membrane bilayer thickness. We conclude that the CpxA could sense changes in the lipid bilayer properties.

Keyword
CpxA envelope stress sensor, lipid bilayer stress, lipid-engineered E. coli strains, surface charge, lateral stress, membrane curvature, bilayer thickness
National Category
Natural Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-102676 (URN)
Funder
Swedish Research Council
Available from: 2014-04-14 Created: 2014-04-14 Last updated: 2014-04-15

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