RESUMO
Plastoglobules (PG) are lipid droplets in chloroplasts and other plastid types having important functions in lipid metabolism. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the PG surface but also to various extents at the thylakoid membrane. PGLs are thought to have structural functions in PG formation and maintenance. The targeting of an Arabidopsis PGL (PGL34) to PG required the full protein sequence with the exception of a short C-terminal stretch. This indicated that PGL targeting relies on correct folding rather than a discrete sequence. PGLs lack strongly hydrophic regions and may therefore extrinsically associate with PG and thylakoid membranes via interaction with hydrophilic headgroups of surface lipids. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 (human immunodeficiency virus capsid particle p24) or HCV (hepatitis C virus core protein) in transplastomic tobacco. A PGL35-HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. The findings indicate that transplastomic PGL35-HIVp24 folded correctly after its synthesis inside the chloroplast and then dually targeted to plastoglobules as well as thylakoid membranes.
Assuntos
Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Genoma de Planta , Proteína do Núcleo p24 do HIV/genética , Proteína do Núcleo p24 do HIV/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Metabolismo dos Lipídeos , Microscopia Eletrônica de Transmissão , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tilacoides/metabolismo , Tilacoides/ultraestrutura , Nicotiana/genética , Nicotiana/ultraestrutura , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismoRESUMO
Plastoglobules are particles specifically located inside different types of plastids. They mainly contain lipids and proteins and are physically attached to thylakoids. Proteomic studies have underlined the role of plastoglobules in diverse plastid metabolic pathways, such as those producing vitamin K, vitamin E, and carotenoids, and have implicated them in plant response to stress. This chapter describes the isolation of pure and intact plastoglobules from Arabidopsis leaves. The procedure starts with the isolation of intact chloroplasts by centrifugation on a Percoll gradient. Plastoglobules are then separated from the plastid membranes by flotation on a sucrose gradient. Finally, the purity of the plastoglobule fraction is verified by immunoblotting.