Faculté des sciences

Plastoglobules : a new destination for recombinant proteins produced in transplastomic plants and characterization of plastidial At-SOUL heme binding protein

Venkatasalam, Shanmugabalaji ; Kessler, Felix (Dir.)

Thèse de doctorat : Université de Neuchâtel, 2012.

Plastoglobules (PG) are lipid droplets existing in most types of plastids. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the surface of PG, but to various extents also at the thylakoid membrane. PGLs are thought to have a structural function in PG, and it is known that almost the complete protein is required for PGLs to assemble on PG. Here,... Di più

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    Summary
    Plastoglobules (PG) are lipid droplets existing in most types of plastids. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the surface of PG, but to various extents also at the thylakoid membrane. PGLs are thought to have a structural function in PG, and it is known that almost the complete protein is required for PGLs to assemble on PG. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 in transplastomic tobacco. A PGL35-HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. These findings suggest that PGL35-HIVp24 folds correctly after its synthesis inside the chloroplast and is then assembled not only on plastoglobules but also on thylakoid membranes. The fusion protein accumulated up to a 1% of the total protein and could be purified by biotin affinity chromatography of a total membrane extract. Targeting of PGL35 fusion proteins to plastoglobules has the potential to become an interesting expression system but it will be necessary to master the parameters that govern the partioning between plastoglobules and thylakoid membranes for this to become an effective technique. This study represents a step forward in this direction.
    In addition to lipids and PGLs, the PG harbours other proteins: The PG proteome in Arabidopsis consist of about two dozen proteins including uncharacterized plastoglobule protein AtSOUL4, which has a predicted heme-binding motif. The Arabidopsis genome contains six proteins with a SOUL motif; two of them AtSOUL4 and AtSOUL5 are predicted to be plastid localized and AtSOUL5 was found in the thylakoid proteome. A SOUL homolog, AtSOUL1, is highly expressed and the soul1 mutant is hypersensitive to red light. In addition, C. reinhardtii eyespot phosphoproteins contained SOUL3. However, due to the presence of SOUL-heme binding proteins in light perception systems and their implication in plant photomorphogenesis, the SOUL heme binding proteins may be involved in light signalling. The heterologously expressed recombinant AtSOUL4 protein binds to heme in vitro as well as in vivo. Moreover, AtPGL35 and AtSOUL4 fluorescent fusion proteins colocalized in chloroplasts. Furthermore, immunoblot analysis of chloroplast membrane fractions shows that AtSOUL4 localized at the plastoglobule. AtSOUL4 has a highly conserved CKII phosphorylation site in C-terminal. Moreover, In vitro phosphorylation studies revealed that AtSOUL4 is phosphorylated most likely by a stromal CKII like kinase. The double knock out of AtSOUL4 and AtSOUL5 revealed a conditional shade avoidance behavior, indicating that AtSOUL4 heme-binding protein might also be involved in the light signalling.