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Endomembrane proton pumps: connecting membrane and vesicle transport Karin Schumacher pH-homeostasis in the endomembrane system requires the activity of proton-pumps. In animals, the progressive acidification of compartments along the endocytic and secretory pathways is critical for protein sorting and vesicle trafficking, and is achieved by the activity of the vacuolar H+-ATPase (V-ATPase). Plants have an additional endomembrane pump, the vacuolar H+-pyrophosphatase (V-PPase), and previous research was largely focused on the respective functions of the two pumps in secondary active transport across the tonoplast. Recent approaches, including reverse genetics, have not only provided evidence that both enzymes play unique and essential roles but have also highlighted the important functions of the two proton pumps in endocytic and secretory trafficking. Addresses ZMBP-Plant Physiology, Universität Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Germany Corresponding author: Schumacher, Karin ([email protected]) Current Opinion in Plant Biology 2006, 9:595–600 This review comes from a themed issue on Cell biology Edited by Laurie G Smith and Ulrike Mayer Available online 27th September 2006 1369-5266/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2006.09.001 Introduction Compartmentation allows the simultaneous occurrence of biochemical processes in different reaction spaces and necessitates the exchange of material via membrane transport or vesicular intermediates. The compartments of the highly dynamic eukaryotic endomembrane system are acidified to varying degrees by the activity of the vacuolar H+-ATPase (V-ATPase). The pH-differential between the cytosol and the lumina is a crucial parameter as it not only affects most biochemical reactions but also enables secondary active transport. In addition to the V-ATPase, plants possess a second endomembrane proton pump, the vacuolar H+-pyrophosphatase (V-PPase). V-ATPase and V-PPase represent up to 30% of total tonoplast protein, and it is therefore not surprising that to date their best-studied function is to maintain ion and metabolite homeostasis by energizing secondary active www.sciencedirect.com transport across the tonoplast [1,2]. The purpose of this review is to highlight recent results demonstrating that both ‘vacuolar’ proton-pumps have important and nonredundant functions in secretory and endocytic trafficking. V-ATPase deficient-mutants V-ATPases constitute a family of highly conserved, multisubunit proton-pumps that are found throughout the endomembrane systems of all eukaryotes [3]. They share a common ancestor with F-ATPases and, like their distant relatives, are composed of two subcomplexes. The peripheral V1 complex, which consists of eight different subunits (subunits A–H), is responsible for ATP hydrolysis; whereas the membrane-integral V0 complex comprises six different subunits (subunits a, c, c0 , c00 , d and e) and is responsible for proton translocation [4]. Yeast vacuolar membrane ATPase (Vma) mutants display a conditional-lethal phenotype, which has greatly facilitated the analysis of V-ATPase subunit composition [5]. Vma-mutants fail to grow at neutral pH, but surprisingly the identity of the cellular organelle whose acidification is essential is still not known. The fact that other yeast mutants that have defects in vacuolar acidification are viable at neutral pH argues, however, that it is not the vacuole [6]. In contrast to the conditional lethality of yeast mutants, mutants identified in several multicellular organisms have shown that V-ATPase activity is essential for embryonic or larval development [7–9]. The underlying cellular defects have only been analyzed in mouse embryos where the lack of the proteolipid subunit leads to the swelling and vacuolation of the Golgi apparatus [10]. The analysis of null-alleles for Arabidopsis genes that encode V-ATPase subunits (VHA) has provided evidence that the V-ATPase is essential, despite the presence of the V-PPase. A T-DNA insertion allele of VHA-A, the single-copy gene that encodes the catalytic subunit of the Arabidopsis V-ATPase, causes complete male and partial female gametophytic lethality [11]. At the cellular level, severe changes in the morphology of Golgi stacks and Golgi-derived vesicles but not of vacuoles were observed in vha-A mutant pollen [11]. Embryos that lack VHA-E1, one of the three isoforms of the VHA-E subunit of V-ATPase, are not viable and display a range of defects including multinucleate cells, cell wall stubs and abnormal division planes that are characteristic of cytokinesis-defective mutants [12]. Like vha-A, vha-E1(tuff)-mutants display an abnormal Current Opinion in Plant Biology 2006, 9:595–600 596 Cell biology organization of Golgi-stacks, indicating that the V-ATPase is required to maintain a functional secretory system during pollen and embryo development [12]. subunit VHA-A of carrot [20], but a recent study of the two highly homologous isoforms of tomato demonstrates that they are expressed in a tissue-specific manner [21]. Plants that have reduced V-ATPase activity, caused either by a weak allele [13] or by RNA interference (RNAi) [14] are viable, but show reduced growth and disturbed ion homeostasis [14,15]. Given the effects on the Golgi apparatus observed in the null mutants, it seems possible that the growth reduction is caused by problems in secretory trafficking rather than by reduced turgor pressure resulting from a lack of osmolyte transport into the vacuole. Further support for this notion comes from the analysis of plants lacking activity of the vacuolar H+/ Ca2+-antiporters of the cation exchanger family (CAX). In cax1 mutants [16] and cax1 cax3 double mutants [17], V-ATPase activity at the tonoplast is, for reasons that remain to be determined, reduced to levels comparable those found in the det3 mutant, which carries a weak allele of VHA-C [13]. The phenotype of det3 is, however, much more severe, indicating that V-ATPase activity in a nonvacuolar compartment is largely responsible for the defects in growth. In yeast, the two isoforms of subunit a, VPH1 (Vacuolar acidification-defective 1) and STV1 (Similar To VPH) [22,23] are differentially localized. Vph1p resides at the tonoplast, whereas Stv1p cycles continuously between a late Golgi compartment and prevacuolar endosomes [23,24]. Two recent publications have addressed the subcellular localization of VHA-a isoforms. Using antibodies raised against one of the VHA-a isoforms of Mesembryanthemum, Kluge et al. [25] demonstrate convincingly that VHAa is an integral part of V-ATPase complexes found in tonoplast-enriched vesicles. Nevertheless, the same antibody labels the endoplasmic reticulum (ER) but fails to detect tonoplast V-ATPase complexes in maize root cells. On this basis, it is argued that an ER-specific VHA-a isoform exists in maize [25]. A systematic analysis of the subcellular localization of the three Arabidopsis VHA-a isoforms, using green fluorescent protein (GFP)-fusion proteins expressed under the endogenous promoters, did not detect an ER-specific isoform. Instead, the distribution of VHA-a isoforms in Arabidopsis is very similar to that in yeast: VHA-a2 and VHA-a3 are found at the tonoplast, whereas VHA-a1 is localized in the trans-Golgi network (TGN) ([26]; see Figure 1). As in yeast Stv1p, the targeting information for TGN localization resides in the amino-terminal cytosolic domain [26]. Moreover, the two yeast isoforms confer different stability and kinetic properties to the holocomplex [24]. The function and regulation of the V-ATPase in the TGN are of particular interest as the use of VHA-a1 as a marker for this compartment has revealed that it is not only the central sorting station of the secretory pathway but also that it functions as an early endosome [26]. V-ATPase isoforms As null alleles are lethal and weak alleles cause pleiotropic phenotypes, more specific approaches are needed to study the biological function of the V-ATPase in different compartments, tissues, developmental stages or stress situations. In Arabidopsis, as in other higher eukaryotes, most V-ATPase subunits are encoded by small gene families [18]. How this vast potential to form V-ATPase complexes with different kinetic and regulatory properties is exploited is only beginning to become apparent. In contrast to the null-allele of the single-copy gene VHAA, which causes male gametophytic lethality, a loss of VHA-E1 is only manifested during embryo development. As both VHA-E2 and VHA-E3 are expressed in developing pollen [12], it seems likely that they act redundantly during this stage of development. During embryogenesis, however, VHA-E1 is the predominant isoform, whereas VHA-E3 is expressed mainly in the endosperm and surrounding maternal tissues, and VHA-E2 is pollen-specific. Interestingly, V-ATPases that differ only in the presence of two isoforms of VHA-E have been isolated from pea epicotyls and showed differences in the kinetics of ATP hydrolysis [19]. Unlike the VHA-E isoforms, which show considerable divergence in protein sequence, isoforms of VHA-c are so similar that differences in targeting or activity changes seem impossible. However, promoter::b-glucuronidase (GUS) analysis revealed that VHA-c1 is highly expressed in expanding tissues whereas VHA-c3 is restricted to tissues that have high demand for V-ATPase, possibly because of particularly active exocytosis [14]. The presence of organellespecific isoforms was first suggested for the catalytic Current Opinion in Plant Biology 2006, 9:595–600 V-ATPase inhibitors The identification of organelle-specific V-ATPase isoforms now allows us to address their functions in the different compartments more specifically but, to date, most of our knowledge of V-ATPase functions in trafficking is based on pharmacology. Concanamycin A (ConcA) and Bafilomycin A (BafA) are membrane-permeable macrolide antibiotics that bind to subunits c [27,28] and a [29], thus inhibiting proton transport by V-ATPases. Numerous pharmacological studies using these potent and specific inhibitors in mammalian cells have established that V-ATPase activity is crucial for many aspects of the mammalian secretory and endocytic pathways, including the dissociation of receptor–ligand complexes [30], the recruitment of proteins that are involved in vesicle formation [31,32], and transport between different endosomal compartments [33–35]. Only a few studies have described the effects of ConcA or BafA on trafficking in plants. In tobacco cells, inhibition www.sciencedirect.com Endomembrane proton pumps Schumacher 597 Figure 1 Proton pumps in the plant endomembrane system. A schematic depiction of the distribution of proton pumps in the plant endomembrane system. Recent findings by Dettmer et al. [26] suggest that endocytic and secretory trafficking merge in the TGN/early endosome, highlighting the importance of this compartment as the central hub of protein trafficking. Question marks indicate that localization of the V-PPase isoforms needs to be identified. PVC: prevacuolar compartment; MVB: multivesicular body. of the V-ATPase interferes with secretion and leads to the mis-sorting of vacuolar proteins, suggesting that the functionality of a non-vacuolar compartment depends on V-ATPase function [36]. It has never been shown that ConcA induces an alkalinization of plant vacuoles. Nevertheless, several recent studies have ascribed the ConcAinduced stabilization of GFP [37] or autophagic bodies in the vacuolar lumen to an increase in vacuolar pH [38,39] rather than to a defect in the sorting of vacuolar hydrolases. Electron microscopy of chemically fixed BY2 cells showed that both ConcA and BafA cause massive vacuolation of the Golgi apparatus [40]. High-pressure-frozen Arabidopsis root cells that were treated with ConcA showed changes in Golgi morphology and aggregations of vesicles [26], similar to those observed in vha-mutant cells. In support of the proposed dual function of the TGN, ConcA blocks both the trafficking of newly synthesized proteins to the PM and transport of endocytic tracers from the PM to the vacuole. How does a lack of V-ATPase activity lead to a block in vesicle trafficking? It is possible that the structural integrity of the Golgi apparatus requires acidification [41], but a direct connection between acidification and vesicle trafficking is now becoming apparent. Recruitment of components of the vesicle budding machinery to membranes can depend on www.sciencedirect.com luminal acidification [31,32,42], but we did not know how the pH information is transmitted across the membrane. Recently, Hurtado-Lorenzo et al. [43] have provided very convincing evidence that subunits of the mammalian V-ATPase interact directly, and in a luminalpH-dependent manner, with proteins that regulate endocytic trafficking. The V-ATPase can thus be regarded as a pH-sensor that is able to communicate luminal pH to cytosolic proteins. Moreover, a function for Vo subunits in membrane fusion, which was first demonstrated for homotypic vacuole fusion in yeast [44], has recently also been identified during synaptic vesicle fusion in Drosophila [45], indicating that this could be a common feature of vesicle-fusion events. The V-PPase In comparison to the V-ATPase, the V-PPase is a much simpler enzyme. It is a homodimer of a single polypeptide and it uses the energy of the phosphoanhydride bond of pyrophosphate (PPi) to drive proton transport across membranes [46]. PPi is a by-product of several biosynthetic processes, and it has therefore been argued that the V-PPase is the predominant proton pump in the vacuoles of young, growing cells [47]. Like the V-ATPase, it has long been known that the V-PPase is not restricted to the tonoplast but is also present and active at the PM, the TGN and in multivesicular bodies [48]. In fact, Current Opinion in Plant Biology 2006, 9:595–600 598 Cell biology Arabidopsis VACUOLAR H+-PYROPHOSPHATASE2 (AVP2)/AVPL1, a representative of the K+-independent type II H+-PPases, is found exclusively in Golgi stacks [49]. beyond proton-pumping is accumulating in other systems, and it will be of great interest to see if plant V-ATPases are also more than just ‘simple’ proton pumps. Acknowledgements A null-allele of AVP1 causes a severe phenotype that is reminiscent of seedlings treated with inhibitors of polar auxin transport [50]. Indeed, levels of the auxin-efflux carrier PIN-FORMED1 (PIN1) and consequently polar auxin transport are reduced in avp1 mutant roots. Furthermore, Li et al. [50] showed that the levels and activity of the plasma membrane P-type H+-ATPase are reduced in these mutants, resulting in an increase in apoplastic pH. Overexpression of AVP1 causes opposite, yet more pronounced, effects on apoplastic pH but, unexpectedly, does not increase levels of PIN1. According to the acid-growth theory, a change in apoplastic pH would directly affect cell expansion and could impinge on the uptake component of polar auxin transport. However, the causal relation between V-PPase levels, the abundance and activity of the plasma membrane H+-ATPase, and the abundance and activity of PIN1 still remains to be elucidated. Manipulation of AVP1 expression not only reveals a close connection between endomembrane proton pumps and auxin-mediated development but might also represent an efficient approach for the engineering of drought-tolerant crops. Overexpression of AVP1 confers tolerance towards water stress in both Arabidopsis [51] and tomato [52]. Initially this effect was ascribed to the ‘classic’ function of the V-PPase in vacuolar uptake of ions, causing increased water uptake. In the light of the recent results, however, it seems likely to be caused by auxin-enhanced root growth. I would like to thank members of my laboratory for many discussions and Felicity de Courcy for critical reading of the manuscript. Work in my laboratory is funded by the German Research Council through grants within SPP1108 and SFB446. The avp1 phenotype clearly indicates that the V-PPase cannot be considered as a mere back-up for the V-ATPase that is only important under conditions in which ATP becomes limiting. Yet the functional relation between the two pumps needs further investigation because several results indicate that they are co-regulated and might interact physically [53]. Most recently, it was shown that Arabidopsis plants that overexpress a V-PPase from the halophyte Suaeda salsa show increased V-ATPase activity under salt and drought stress [54]. Conclusions Although it has been known for a long time that the V-ATPase and the V-PPase are found throughout the endomembrane system, our view of their biological function was dominated by their role in energizing transport across the tonoplast. In recent years, our view has been substantially widened and it is now clear that they are also important players in trafficking vesicles along the endocytic and secretory pathways. The next challenge is to understand how the two proton-pumps contribute to the dynamics of the endomembrane system. Moreover, evidence for functions of the V-ATPase Current Opinion in Plant Biology 2006, 9:595–600 References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Sze H, Li X, Palmgren MG: Energization of plant cell membranes by H+-pumping ATPases. Regulation and biosynthesis. Plant Cell 1999, 11:677-690. 2. Kluge C, Lahr J, Hanitzsch M, Bolte S, Golldack D, Dietz KJ: New insight into the structure and regulation of the plant vacuolar H+-ATPase. J Bioenerg Biomembr 2003, 35:377-388. 3. Nishi T, Forgac M: The vacuolar (H+)-ATPases — nature’s most versatile proton pumps. Nat Rev Mol Cell Biol 2002, 3:94-103. 4. Beyenbach KW, Wieczorek H: The V-type H+ ATPase: molecular structure and function, physiological roles and regulation. J Exp Biol 2006, 209:577-589. 5. Kane PM: The where, when, and how of organelle acidification by the yeast vacuolar H+-ATPase. 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Planta 1999, 208:205-211. 49. Mitsuda N, Enami K, Nakata M, Takeyasu K, Sato MH: Novel type Arabidopsis thaliana H+-PPase is localized to the Golgi apparatus. FEBS Lett 2001, 488:29-33. 50. Li J, Yang H, Peer WA, Richter G, Blakeslee J, Bandyopadhyay A, Titapiwantakun B, Undurraga S, Khodakovskaya M, Richards EL et al.: Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development. Science 2005, 310:121-125. This article establishes a connection between the levels of the V-PPase AVP1, the PM H+-ATPase and the auxin efflux carrier PIN1. Although the causality of the observed effects remains to be elucidated, it provides striking evidence of the importance of endomembrane proton-pumps in trafficking of PM proteins. Current Opinion in Plant Biology 2006, 9:595–600 51. Gaxiola RA, Li J, Undurraga S, Dang LM, Allen GJ, Alper SL, Fink GR: Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proc Natl Acad Sci USA 2001, 98:11444-11449. 52. Park S, Li J, Pittman JK, Berkowitz GA, Yang H, Undurraga S, Morris J, Hirschi KD, Gaxiola RA: Up-regulation of a H+-pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistant crop plants. Proc Natl Acad Sci USA 2005, 102:18830-18835. The authors show that overexpression of the V-PPase might provide a general strategy to create drought-tolerant crop plants. 53. Fischer-Schliebs E, Mariaux JB, Luettge U: Stimulation of H+-transport activity of vacuolar H+-ATPase by activation of H+-PPase in Kalanchoe blossfeldiana. Biol Plant 1997, 39:169-177. 54. Guo S, Yin H, Zhang X, Zhao F, Li P, Chen S, Zhao Y, Zhang H: Molecular cloning and characterization of a vacuolar H+-pyrophosphatase gene, SsVP, from the halophyte Suaeda salsa and its overexpression increases salt and drought tolerance of Arabidopsis. Plant Mol Biol 2006, 60:41-50. Interestingly, this paper shows that overexpression of the V-PPase might also lead to enhanced activity of the V-ATPase. www.sciencedirect.com