RESUMO
Vacuoles are multifunctional organelles essential for the sessile lifestyle of plants. Despite their central functions in cell growth, storage, and detoxification, knowledge about mechanisms underlying their biogenesis and associated protein trafficking pathways remains limited. Here, we show that in meristematic cells of the Arabidopsis thaliana root, biogenesis of vacuoles as well as the trafficking of sterols and of two major tonoplast proteins, the vacuolar H(+)-pyrophosphatase and the vacuolar H(+)-adenosinetriphosphatase, occurs independently of endoplasmic reticulum (ER)-Golgi and post-Golgi trafficking. Instead, both pumps are found in provacuoles that structurally resemble autophagosomes but are not formed by the core autophagy machinery. Taken together, our results suggest that vacuole biogenesis and trafficking of tonoplast proteins and lipids can occur directly from the ER independent of Golgi function.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Retículo Endoplasmático/metabolismo , Vacúolos/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Genes Reporter , Complexo de Golgi/metabolismo , Concentração de Íons de Hidrogênio , Pirofosfatase Inorgânica/genética , Pirofosfatase Inorgânica/metabolismo , Metabolismo dos Lipídeos , Meristema/enzimologia , Meristema/genética , Meristema/fisiologia , Meristema/ultraestrutura , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Raízes de Plantas/ultraestrutura , Plantas Geneticamente Modificadas , Transporte Proteico , Proteínas Recombinantes de Fusão , Esteróis/metabolismoRESUMO
How individual protein subunits assemble into the higher order structure of a protein complex is not well understood. Four proteins dedicated to the assembly of the V(0) subcomplex of the V-adenosine triphosphatase (V-ATPase) in the endoplasmic reticulum (ER) have been identified in yeast, but their precise mode of molecular action remains to be identified. In contrast to the highly conserved subunits of the V-ATPase, orthologs of the yeast assembly factors are not easily identified based on sequence similarity. We show in this study that two ER-localized Arabidopsis proteins that share only 25% sequence identity with Vma21p can functionally replace this yeast assembly factor. Loss of AtVMA21a function in RNA interference seedlings caused impaired cell expansion and changes in Golgi morphology characteristic for plants with reduced V-ATPase activity, and we therefore conclude that AtVMA21a is the first V-ATPase assembly factor identified in a multicellular eukaryote. Moreover, VMA21p acts as a dedicated ER escort chaperone, a class of substrate-specific accessory proteins so far not identified in higher plants.