RESUMEN
Deg proteases are involved in protein quality control in prokaryotes. Of the three Arabidopsis (Arabidopsis thaliana) homologs, Deg1, Deg5, and Deg8, located in the thylakoid lumen, Deg1 forms a homohexamer, whereas Deg5 and Deg8 form a heterocomplex. Both Deg1 and Deg5-Deg8 were shown separately to degrade photosynthetic proteins during photoinhibition. To investigate whether Deg1 and Deg5-Deg8 are redundant, a full set of Arabidopsis Deg knockout mutants were generated and their phenotypes were compared. Under all conditions tested, deg1 mutants were affected more than the wild type and deg5 and deg8 mutants. Moreover, overexpression of Deg5-Deg8 could only partially compensate for the loss of Deg1. Comparative proteomics of deg1 mutants revealed moderate up-regulation of thylakoid proteins involved in photoprotection, assembly, repair, and housekeeping and down-regulation of those that form photosynthetic complexes. Quantification of protein levels in the wild type revealed that Deg1 was 2-fold more abundant than Deg5-Deg8. Moreover, recombinant Deg1 displayed higher in vitro proteolytic activity. Affinity enrichment assays revealed that Deg1 was precipitated with very few interacting proteins, whereas Deg5-Deg8 was associated with a number of thylakoid proteins, including D1, OECs, LHCBs, Cyt b 6 f, and NDH subunits, thus implying that Deg5-Deg8 is capable of binding substrates but is unable to degrade them efficiently. This work suggests that differences in protein abundance and proteolytic activity underlie the differential importance of Deg1 and Deg5-Deg8 protease complexes observed in vivo.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteostasis , Serina Endopeptidasas/metabolismo , Tilacoides/enzimología , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Mutación , Fenotipo , Fotosíntesis , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Proteómica , Plantones/enzimología , Plantones/genética , Plantones/fisiología , Serina Endopeptidasas/genética , Tilacoides/fisiologíaRESUMEN
Rhomboids are intra-membrane serine proteases whose sequences are found in nearly all organisms. They are involved in a variety of biological functions in both eukaryotes and prokaryotes. Localization assays revealed that two Arabidopsis thaliana rhomboid-like proteases (AtRBL), AtRBL8 and AtRBL9, are targeted to the chloroplast. Using transgenic plants expressing epitope-tagged AtRBL9, we localized AtRBL9 to the chloroplast inner envelope membrane, with both its N- and C-termini facing the stroma. Mass spectrometry analyses confirmed this localization, and suggested that this is also the case for AtRBL8. Both are proteins of very low abundance. The results of size-exclusion chromatography implied that AtRBL9 forms homo-oligomers. In search of a putative function, a comparative proteomic analysis was performed on wild-type and double-knockout plants, lacking both AtRBL8 and AtRBL9, using the iTRAQ method. Of 180 envelope proteins, the level of only a few was either increased or decreased in the mutant line. One of the latter, allene oxide synthase, is involved in jasmonic acid biosynthesis. This observation provides an explanation for the recently reported aberration in flower morphology that is associated with the loss of AtRBL8.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cloroplastos/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Membranas Intracelulares/enzimología , Oxidorreductasas Intramoleculares/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Permeabilidad de la Membrana Celular , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/genética , Cromatografía en Gel , Proteínas Cromosómicas no Histona/genética , Clonación Molecular , Secuencia Conservada , Ciclopentanos/metabolismo , Técnicas de Inactivación de Genes , Genes de Plantas , Membranas Intracelulares/metabolismo , Oxidorreductasas Intramoleculares/genética , Espectrometría de Masas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oxilipinas/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Proteoma/análisis , Proteoma/metabolismoRESUMEN
How can cells sense their own size to coordinate biosynthesis and metabolism with their growth needs? We recently proposed a motor-dependent bidirectional transport mechanism for axon length and cell size sensing, but the nature of the motor-transported size signals remained elusive. Here, we show that motor-dependent mRNA localization regulates neuronal growth and cycling cell size. We found that the RNA-binding protein nucleolin is associated with importin ß1 mRNA in axons. Perturbation of nucleolin association with kinesins reduces its levels in axons, with a concomitant reduction in axonal importin ß1 mRNA and protein levels. Strikingly, subcellular sequestration of nucleolin or importin ß1 enhances axonal growth and causes a subcellular shift in protein synthesis. Similar findings were obtained in fibroblasts. Thus, subcellular mRNA localization regulates size and growth in both neurons and cycling cells.