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Basic clusters and amphipathic helices contribute to interactions of Myr1/Syh1 with membrane phospholipids
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Åke Wieslander)
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]

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: urn:nbn:se:su:diva-62030OAI: oai:DiVA.org:su-62030DiVA: diva2:439464
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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