The Endoplasmic Reticulum Is the Main Membrane Source for Biogenesis of the Lytic Vacuole in Arabidopsis W Corrado Viotti, a,b,1 Falco Krüger, a,1 Melanie Krebs, a Christoph Neubert, a Fabian Fink, a Upendo Lupanga, a David Scheuring, a Yohann Boutté, b,2 Márcia Frescatada-Rosa, b Susanne Wolfenstetter, c Norbert Sauer, c Stefan Hillmer, a Markus Grebe, b and Karin Schumacher a,3 a Centre for Organismal Studies, Plant Developmental Biology, University of Heidelberg, 69120 Heidelberg, Germany b Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 90187 Umea, Sweden c Molecular Plant Physiology, University of Erlangen-Nürnberg, 91058 Erlangen, Germany 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. INTRODUCTION The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell. Vacuoles fulfill multiple functions that are essential for the lifestyle of plants. They are the main store for solutes and serve, through osmotic water uptake, as a hydrostatic skeleton that in combination with the cell wall provides the driving force underlying cell growth and reversible changes in cell volume. The success of land plants as sessile organisms is directly linked to the vacuole as it enables plant cells to buffer changes in the availability of essential nutrients, to detoxify the cytosol when challenged by harmful molecules, and to defend themselves against biotic challenges. Moreover, most plant vacuoles serve as lysosomes in which proteins delivered either by endocytosis or autophagy are digested by hydrolytic enzymes. Based on this function, plant vacuoles are classified as either lytic vacuoles (LVs) or protein storage vacuoles (PSVs) typically found in seeds (Matile, 1978; Marty, 1999; Jiang et al., 2000). All functions of LVs in cellular homeostasis require massive fluxes of molecules across the tonoplast, the membrane sepa- rating cytosol and vacuolar lumen. Vacuolar transport is chan- neled by a battery of transport proteins that are energized by the combined activity of two proton pumps, the vacuolar H + - pyrophosphatase (V-PPase) and the vacuolar H + -adenosine- triphosphatase (V-ATPase). The combined activity of the two pumps is assumed to not only create the proton gradient and the membrane potential necessary to transport compounds against their concentration or electrochemical gradient, but also to maintain the acidic pH required for the lytic function (Maeshima, 2001; Gaxiola et al., 2007). V-ATPases are highly conserved, multisubunit proton pumps that consist of two subcomplexes, the peripheral V 1 complex responsible for ATP hydrolysis and the membrane-integral V o complex responsible for proton translocation (Matile, 1978; Marty, 1999; Jiang et al., 2000; Sze et al., 2002). In comparison, the V-PPase, a homo- dimer of a single polypeptide, is a much simpler enzyme that uses PPi to pump proton transport across the tonoplast (Maeshima, 2001; Gaxiola et al., 2007). Due to their different energy sources, it is generally assumed that the combined action of the two enzymes enables plants to maintain transport into the vacuole even under stressful conditions (Martinoia et al., 2007). Indeed, recent analysis of mutants has confirmed that the V-ATPase is of general importance for vacuolar transport (Krebs et al., 2010), whereas the role of the V-PPase, at least under nonstress conditions, seems to be limited to cytosolic PPi ho- meostasis (Ferjani et al., 2011). Both proton pumps are among the most abundant tonoplast proteins (Carter et al., 2004; Jaquinod et al., 2007; Schulze et al., 2012); however, the molecular mechanisms underlying their sorting and targeting remains to be identified. Assembly of the V o subcomplex of the V-ATPase takes place in the endoplasmic reticulum (ER) and requires the presence of dedicated assembly factors (Neubert et al., 2008). We have shown previously that the subcellular distribution of the V-ATPase is determined by the isoform of the membrane- integral subunit VHA-a. Incorporation of VHA-a1 targets the complex to the trans-Golgi network (TGN)/early endosome (EE), 1 These authors contributed equally to this work. 2 Current address: Laboratoire de Biogenèse Membranaire, University of Bordeaux 2, 33076 Bordeaux, France. 3 Address correspondence to karin.schumacher@cos.uni-heidelberg.de. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Karin Schumacher (karin. schumacher@cos.uni-heidelberg.de). W Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.113.114827 The Plant Cell, Vol. 25: 3434–3449, September 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.