000030458 001__ 30458
000030458 005__ 20130211180923.0
000030458 0247_ $$2urn$$aurn:nbn:ch:rero-004-111168
000030458 0248_ $$aoai:doc.rero.ch:20121010115155-BK$$pthesis$$pthesis_urn$$zcdu34$$zcdu57$$zreport$$zbook$$zjournal$$zcdu16$$zpostprint$$zpreprint$$zcdu1$$zdissertation$$zunine
000030458 035__ $$aR007157137
000030458 041__ $$aeng
000030458 080__ $$a57
000030458 100__ $$aVenkatasalam, Shanmugabalaji
000030458 245__ $$9eng$$aPlastoglobules$$ba new destination for recombinant proteins produced in transplastomic plants and characterization of plastidial At-SOUL heme binding protein
000030458 502__ $$92012$$aThèse de doctorat : Université de Neuchâtel, 2012
000030458 506__ $$ffree
000030458 520__ $$9eng$$aPlastoglobules (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.  <br> 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 <i>soul1</i> mutant is hypersensitive to red light. In addition, <i>C. reinhardtii</i> 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 <i>in vitro</i> as well as <i>in vivo</i>. 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, <i>In vitro</i> 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.
000030458 600__ $$2rero$$9650_7$$aChloroplastes
000030458 600__ $$2rero$$9650_7$$aCellules végétales
000030458 600__ $$2rero$$9650_7$$aBiologie moléculaire végétale
000030458 695__ $$9eng$$achloroplast ; plastoglobule ; plastoglobulin ; plastid transformation ; molecular farming ; HIVp24 ; HCV core protein ; <i>Nicotiana tabacum</i> ; SOUL heme binding protein ; tetrapyrrole
000030458 700__ $$aKessler, Felix$$eDir.
000030458 8564_ $$f00002281.pdf$$qapplication/pdf$$s3434213$$uhttp://doc.rero.ch/record/30458/files/00002281.pdf$$yorder:1$$zTexte intégral
000030458 918__ $$aFaculté des sciences$$bRue Emile-Argand 11, 2007 Neuchâtel
000030458 919__ $$aUniversité de Neuchâtel$$bNeuchâtel$$ddoc.support@rero.ch
000030458 980__ $$aTHESIS$$bUNINE$$fTH_PHD
000030458 990__ $$a20121010115155-BK