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1.
PLoS One ; 8(9): e74421, 2013.
Article in English | MEDLINE | ID: mdl-24040242

ABSTRACT

In most organisms, storage lipids are packaged into specialized structures called lipid droplets. These contain a core of neutral lipids surrounded by a monolayer of phospholipids, and various proteins which vary depending on the species. Hydrophobic structural proteins stabilize the interface between the lipid core and aqueous cellular environment (perilipin family of proteins, apolipoproteins, oleosins). We developed a genetic approach using heterologous expression in Saccharomyces cerevisiae of the Arabidopsis thaliana lipid droplet oleosin and caleosin proteins AtOle1 and AtClo1. These transformed yeasts overaccumulate lipid droplets, leading to a specific increase in storage lipids. The phenotype of these cells was explored using synchrotron FT-IR microspectroscopy to investigate the dynamics of lipid storage and cellular carbon fluxes reflected as changes in spectral fingerprints. Multivariate statistical analysis of the data showed a clear effect on storage carbohydrates and more specifically, a decrease in glycogen in our modified strains. These observations were confirmed by biochemical quantification of the storage carbohydrates glycogen and trehalose. Our results demonstrate that neutral lipid and storage carbohydrate fluxes are tightly connected and co-regulated.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/chemistry , Calcium-Binding Proteins/metabolism , Lipids/chemistry , Organelles/metabolism , Plant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Gene Expression , Glycogen/metabolism , Multivariate Analysis , Organelles/chemistry , Organelles/ultrastructure , Plant Proteins/chemistry , Plant Proteins/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/ultrastructure , Single-Cell Analysis , Spectroscopy, Fourier Transform Infrared/methods , Synchrotrons , Trehalose/metabolism
2.
Biochim Biophys Acta ; 1828(8): 1881-8, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23603223

ABSTRACT

In cells, from bacteria to plants or mammals, lipids are stored in natural emulsions called oil bodies (OBs). This organelle is surrounded by a phospholipid monolayer which is thought to contain integral proteins involved in its stabilization. The insertion and fold of these proteins into the phospholipid monolayer are poorly understood. In seed OBs, the most abundant integral proteins are oleosins, which contain a 70-residue central hydrophobic domain. The secondary structure of solubilized oleosins varies greatly from mainly alpha helices to a predominantly beta sheets depending on the detergent used. To study the fold of integral membrane proteins inserted in a cellular OB environment, S3 protein, the major Arabidopsis thaliana seed oleosin, was targeted to Saccharomyces cerevisiae OBs. The diameter of purified yeast OBs harboring S3 or S3 fused with the Green Fluorescent Protein (GFP) was smaller and more homogeneous than plant OBs. Comparison of the secondary structure of S3 and S3-GFP was used to validate the structure of folded S3. Circular dichroism using synchrotron radiation indicated that S3 and S3-GFP in yeast OBs contain mainly beta secondary structures. While yeast OBs are chemically different to A. thaliana seed OBs, this approach allowed the secondary structure of S3 in OB particles to be determined for the first time.


Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/metabolism , Cell Membrane/chemistry , Oils/chemistry , Protein Folding , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Arabidopsis Proteins/metabolism , Cell Membrane/metabolism , Circular Dichroism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hydrophobic and Hydrophilic Interactions , Immunoblotting , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Fluorescence , Oils/metabolism , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/chemistry , Protein Structure, Secondary , Recombinant Fusion Proteins/genetics
3.
Biochim Biophys Acta ; 1808(3): 706-16, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21146495

ABSTRACT

Seed lipid bodies constitute natural emulsions stabilized by specialized integral membrane proteins, among which the most abundant are oleosins, followed by the calcium binding caleosin. These proteins exhibit a triblock structure, with a highly hydrophobic central region comprising up to 71 residues. Little is known on their three-dimensional structure. Here we report the solubilization of caleosin and of two oleosins in aqueous solution, using various detergents or original amphiphilic polymers, amphipols. All three proteins, insoluble in water buffers, were maintained soluble either by anionic detergents or amphipols. Neutral detergents were ineffective. In complex with amphipols the oleosins and caleosin contain more beta and less alpha secondary structures than in the SDS detergent, as evaluated by synchrotron radiation circular dichroism. These are the first reported structural results on lipid bodies proteins maintained in solution with amphipols, a promising alternative to notoriously denaturing detergents.


Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis Proteins/metabolism , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Lipids/analysis , Plant Proteins/chemistry , Plant Proteins/metabolism , Protein Folding , Seeds/chemistry , Water/chemistry , Circular Dichroism , Hydrophobic and Hydrophilic Interactions , Polymers/chemistry , Protein Structure, Secondary , Solubility
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