RESUMEN
ATP synthases in chloroplasts (cpATPase) and mitochondria (mtATPase) are responsible for ATP production during photosynthesis and oxidative phosphorylation, respectively. Both enzymes consist of two multisubunit complexes, the membrane-bound coupling factor O and the soluble coupling factor 1. During cpATPase biosynthesis, several accessory factors facilitate subunit production and orchestrate complex assembly. Here, we describe a new auxiliary protein in Arabidopsis thaliana, which is required for cpATPase accumulation. AtCGLD11 (CONSERVED IN THE GREEN LINEAGE AND DIATOMS 11) is a protein without any known functional domain and shows dual localization to chloroplasts and mitochondria. Loss of AtCGLD11 function results in reduced levels of cpATPase and impaired photosynthetic performance with lower rates of ATP synthesis. In yeast two-hybrid experiments, AtCGLD11 interacts with the ß subunits of the cpATPase and mtATPase. Our results suggest that AtCGLD11 functions in F1 assembly during cpATPase biogenesis, while its role in mtATPase biosynthesis may not, or not yet, be essential.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Arabidopsis/metabolismo , ATPasas de Translocación de Protón de Cloroplastos/metabolismo , Proteínas de Arabidopsis/genética , ATPasas de Translocación de Protón de Cloroplastos/genética , Cloroplastos/enzimología , Cloroplastos/metabolismoRESUMEN
Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Transporte Biológico , Retículo Endoplásmico/metabolismo , Fertilidad/genética , Flores/genética , Flores/metabolismo , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Metabolismo de los Lípidos/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plastidios/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Alineación de SecuenciaRESUMEN
All members of the YidC/Oxa1/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that Alb4 is required in assembly and/or stability of the CF1CF0-ATP synthase (ATPase). alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not Alb3 physically interacts with the subunits CF1beta and CF0II. Summarizing, the data indicate that Alb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.