Small CTP-Binding Proteins and Membrane Biogenesis in Plants

Plant Physiol. (1994) 106: 1-6 zyxwvutsrq zyxw zyxwv Small CTP-Binding Proteins and Membrane Biogenesis in Plants' Desh Pal S. Verma*, Choong-lll Cheon', and Zonglie Hong Department of Molecular Genetics and Biotechnology Center, The Ohio State University, Columbus, Ohio 43210 One of the amazing features of the cellular machinery is that a11 organeller and membrane proteins, as well as those destined for secretion, have an attached address label for targeting to a specific site. Following synthesis, these proteins are folded, shipped, delivered, and received at the right compartment. Their assigned functions are performed only when they are properly placed at a designated site in the cell. Membrane vesicles play an essential role in protein transport as carriers of specific proteins to intracellular compartments. This process begins immediately after perception of specific signals and involves membrane ruffling, budding and transport of ER vesicles, fusion and passage through the Golgi, and release of vesicles from trans-Golgi cistemae to target to the vacuoles and plasma membrane. The components of the vesicle-mediatedprotein trafficking system are not, however, well defined. It is not known what kind of biochemical principles are operative for unidirectional transport of vesicles. How are vesicles fused to the target compartment? Although much remains to be understood, many studies from yeast and mammalian systems have identified some key players in this pathway. Isolation of plant homologs of some of these proteins has confmed that these steps are conserved in evolution and must involve well-defined reactions. We focus here on the relevance of small GTP-binding proteins in vesicle-mediated protein transport (for earlier reviews, see Balch, 1990; Bednarek and Raikhel, 1992; Pryer et al., 1992; Terryn et al., 1993; Zerial and Stenmark, 1993). cell proliferation and directing transport of vesicles to their destinations (Boume et al., 1991). Based on the similarity in amino acids and the presumed function, small GTP-binding proteins can be grouped into five subfamilies: Ras/Ras-like, Ran/TC4, Rho/Rac, Rab/Ypt, and Arf/Sar. Ras is the best-characterized small GTP-binding protein. Ras and Ras-like (including Rap, Ral, and Rras) proteins are believed to be involved in signal transduction and regulation of cell growth and differentiation (Hall, 1990). Ran/TC4 (including human TC4 and yeast GSP and SPI) proteins are localized in the nucleus and are required for DNA synthesis and protein import into the nucleus (Moore and Blobel, 1993; Lounsbury et al., 1994). The Rho/Rac family has been implicated in cytoskeletal organization and regulation of growth factor-induced membrane ruffling (Ridley et al., 1992). Members of the Rab/Ypt subfamily have been shown to be involved in vesicular transport. Identification of Rab proteins began with cloning of YPTI, a ras-like gene in yeast, followed by identification and isolation of SEC4 (see Balch [1990] and refs. therein). Disruption of the SEC4 gene results in accumulation of membrane vesicles in yeast, whereas duplication of SEC4 suppresses sec mutants of post-Golgi events, implicating its product in vesicular transport at the post-Golgi level. Sec4p (the protein encoded by SEC4) is localized on the plasma membrane and on secretory vesicles. A mutation in YPTl causes defects in early secretion and membrane proliferation, and the function of Yptlp has been suggested to be in the transport of vesicles from ER to Golgi. Further studies on Yptlp and Sec4p led to the discovery of their homologs, Rab proteins, in both mammalian and plant cells. A number of genes encoding Rab proteins have been isolated. Different Rab proteins are localized on distinct compartments in the secretory and endocytic pathways (see Zerial and Stenmark, 1993). The amino acid sequence of the Arf/Sar proteins suggests that they are distantly related to the Rab/Ypt family. Arf and Sarl subgroups share high homology (60%) with each other zyx zyxwv THE DlVERSlTY O F THE SMALL CTP-BINDINC PROTEIN FAMILY A group of GTP-binding proteins ranging in molecular mass from 20 to 30 kD (referred to as small GTP-binding proteins) is found in a11 eukaryotic cells. These proteins share high amino acid sequence identity and overall structure, suggesting that they evolved from a common ancestral gene. These proteins also share a common mechanism to function as a molecular switch that can be tumed on by binding to GTP and tumed off by hydrolyzing GTP to GDP. This switch enables transduction of signals across membranes, controlling zyxw Abbreviations:azaC, 5-azacytidine; GAP, GTPase-activatingprotein; GDI, GDP dissociation inhibitor; GEF, guanine-nucleotideexchange factor; NSF, N-ethylmaleimide-sensitivefusion protein; PBF, peribacteroid fluid; PBM, peribacteroid membrane; SNAP, soluble NSF attachmentproteins;SNARE, SNAP receptors; TGN, transGol@ network; t-SNARE, SNAP receptors that exist on the target membrane; V-SNARE, SNAP receptors that exist on the vesicular membrane. ' This study was supportedby National Science Foundationgrants DCB 88-19399 and DCB 89-04101. Present address: Plant Gene Expression Center, U.S. Department of Agriculture, Albany, CA 94710. * Correspondingauthor; fax 1-614-292-5379. 1 Downloaded from www.plantphysiol.org on June 7, 2015 - Published by www.plant.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. 2 zyxwvutsrqpo zyx zyxwvut zy zyxwvutsrqp Verma et al. but have distinct functions. Arf (ADP ribosylation factor) was originally identified and purified as a protein cofactor required for ADP ribosylation of the a subunit of heterotrimeric G proteins, proteins that function in signal transduction. Arf is associated with Golgi membranes and is essential in forming coatomers, the protein complexes that coat Golgi-derived vesicles. Arf and other coat proteins of vesicles were suggested to be involved in the vesicle budding process, which is regulated by heterotrimeric G proteins (Bauerfeind and Huttner, 1993). SARl was discovered as a multicopy suppressor of a secl2 temperature-sensitive strain. It interacts with Secl2p (a GEF specific for Sarlp), Sec23p (a GAP specific for Sarlp), and Sec24p. Formation of this complex is required for vesicle budding from the ER (Nakano and Muramatsu, 1989; Barlowe and Schekman, 1993). SMALL CTP-BINDING PROTEINS ARE ASSOCIATED WlTH DIFFERENT MEMBRANE COMPARTMENTS Despite sharing a high leve1 of sequence similarity, small GTP-binding proteins appear to associate with distinct membrane compartments and perform different biological functions. Most of the Ras/Ras-like, Rho/Rac, and Rab/Ypt proteins possess a variable sequence at the C terminus that contains a CAAX, CXC, or CC motif. This motif is a signal for addition of a farnesyl or geranylgeranyl lipid moiety to the proteins, a process known as prenylation by which these proteins are attached to membranes. Many Ras/Ras-like proteins have been localized on the plasma membrane (Hall, 1990). Unlike other small GTP-binding proteins, Ran/TC4 proteins are localized in the nucleus. These proteins lack the consensus CAAX motif for prenylation at the C terminus, but they have an acidic C-terminal tail that has been implicated in reactions with other nuclear proteins (Moore and Blobel, 1993; Lounsbury et al., 1994). Arf and Ar1 (Arf-like)proteins also lack the CAAX motif at the C terminus, but they have a Gly at position 2 of the N terminus that serves as a site for N myristoylation (Bauerfeind and Huttner, 1993). Sarl proteins do not contain motifs for potential membrane modification. These proteins are associated with the ER membranes by forming a complex with Secl2p, an integral ER membrane protein (Nakano and Muramatsu, 1989; Barlowe and Schekman, 1993). The Rab/Ypt subfamily comprises a large number of proteins (at least 30) with distinct subcellular locations (Table I; see also Pryer et al., 1992; Zerial and Stenmark, 1993). Rabla and Rab2 are shown to be involved in the ER-Golgi transport and Rab3 is shown to be involved in transport of synaptic vesicles. Rab4, Rab5, Rab7, Rab9, Rab22, and Rab24 are engaged in the endocytic pathway, whereas both Rab4 and Rab5 are associated with the early endosomes and have different roles in endocytosis (Wichmann et al., 1992; Olkkonen et al., 1993). Rab5 appears to function in endosomeendosome fusion and Rab4 appears to function in a recycling pathway from early endosomes to the cell surface. Vps21, a Rab5 homolog from yeast, is required for the sorting of vacuolar proteins (Horazdovsky et al., 1994). Rab6p is localized in the Golgi and plays a role at an early step in the biogenesis of synaptic vesicles (Tixier-Vida1 et ai., 1993). Rab7p is located on late endosomes and may be required for Plant Physiol. Vol. 106, 1994 transport from early to late endosomes, and Rab9 is involved in transport from late endosomes to the trans-Golgi network. ACCESSORY FACTORS ASSOCIATED WlTH SMALL CTP-BINDING PROTEINS Similar to other GTP-binding proteins, Rab proteins undergo GTP-bound and GDP-bound states. T h i j switch is regulated by several accessory proteins. GAP accekrates GTP hydrolysis by enhancing the intrinsic GTPase activity associated with GTP-binding proteins; otherwise, thv GTP hydrolysis Iate is very low. GEF mediates the replacement of GDP with GTP, causing GTPase to become active (Fig. 1). GDI inhibits the dissociation of GDP and prevents the GDPbound form of Rab proteins from binding to membranes. These accessory proteins were first identified for Ras and heterotrirneric G proteins (Boguski and McCormick, 1993). Recently, Rab-specific accessory proteins have been identified. The GAP protein of Ypt6p stimulates the GTPase activity of Ypt6p but not that of Rab proteins (Strom et al., 1993). Yeast Dss4 protein was found to have GEF activity for Sec4 and its mammalian homolog. Mss4 protein has G EF activity for Yptlp and Rab3a as well as for Sec4p (Burton et al., 1993; Moya et al., 1993).A newly identified membrane component, termed GDI-dissociation factor, has been implicated in recruitment of specific Rab proteins into the vesicles (Soldati et al., 1994). This protein causes dissociation of the Rítb-rabGDI complex and thus promotes binding of the Rab prc tein to the membrane. In addition to Rab proteins and their accessory protein factors, several other essential proteins of the trartsport machinery have been identified. Both mammalian and yeast cells require NSF and SNAPs for vesicle fusion (Rothman and Orci, 1992). Recently, membrane proteins that bind to SNAPs were isolated from brain extract and suggested to mediate the fusion of synaptic vesicles (Sollner et al., 1993). These findings have led to a general model of vesicle transport and fusion, termed the SNARE hypothesis (Fig. 2; see also Novick and Brennwald, 1993; Takizawa and Malhotra, 1993; Zerial and Stenmark, 1993). This model presumes that the SNAP-binding proteins are SNAP receptors that exist on vesicular membrane (v-SNARE) and the target inembrane (t-SNARE). According to the model, V-SNARE c n vesicles may bind specifically to t-SNARE on the target membrane as a first step of fusion and then interact with SNAP. NSF and other unidentified proteins then drive the fusion process. In the context of the SNARE hypothesis, Rab proteins may regulate V-SNARE in a way that V-SNARE woulcl be active only when it is bound to GTP-Rab proteins (Novick and Brennwald, 1993). It remains to be answered whether the SNARE hypothesis can be generally true for the various steps of intracellular transport and is applicable to plant cells. SMALL GTP-BINDING PROTEINS IN PLANTS Early experiments on mammalian systems have demonstrated that GTP-binding proteins, when resolvetl by SDSPAGE and transferred to nitrocellulose filters, retain the ability to bind to GTP. GTPTS, a nonhydrolyzable GTP analog, binds irreversibly to GTP-binding protliins. This Downloaded from www.plantphysiol.org on June 7, 2015 - Published by www.plant.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. zyxw z zyxwvu zyxwvutsr Small GTP-Binding Proteins and Membrane Biogenesis in Plants 3 Localization and possible function of m a / / CTP-binding proteins in different membrane ComDartments Table 1. Protein Ras Ran Localization Plasma mem brane Nucleus Rac Rho ? Golgi, post-Golgi vesicles Golgi Arf Possible Function Signal transduction Nuclear protein import, DNA synthesis Phagocytosis, membrane ruffling Actin cytoskeleton Regulate budding from the ER and fusion at the Golgi stacks, endosomes, and nuclear vesicles Sar Rab Rabla Rabl b Rab2 ER Vesicular budding from t h e E R ER, Golgi ER-Golgi intermediate compart- ER-Golgi transport ER-cis-Golgi transport ER-Golgi transport Rab3a ment Synaptic vesicles, chromaffin Regulated exocytosis Rab3b Rab3c Rab4 granules Mainly in cytosol Synaptic vesicles Early endosomes Rab5 Early endosomes, plasma mem- Rab6 Rab7 Raba Rab9 Rabll ? brane TGN, post-Golgi transport vesicles Late endosomes Post-Golgi basolateral secretory vesicles Late endosomes, TGN TCN, secretory granules, synaptic vesicles Rabl2 Rabl3 Golgi Tight junction Rabl7 Rab22 Rab24 Basolateral plasma membrane Plasma membrane, endosomes ER, cis-Golgi, late endosomes Regulated exocytosis Regulated exocytosis Early endosome-plasma membrane recycling pathway Plasma membrane-early endosome transport, fusion of early endosomes Budding from TGN Transport in endocytic pathway Golgi-plasma membrane transport Late endosome-TGN transport ? ? Polarized transport, assembly of tight junction Transcellular transport Endocytic pathway Endocytic pathway z zyxwvutsrq property has allowed researchers to detect the presence of small GTP-binding proteins in plant extracts and in thylakoid and microsomal membranes (Hasunuma and Funadera, 1987; Zbell et al., 1990). Two major proteins from soybean plasma membranes (24 and 28 kD, Zbell et al., 1990) and from root nodule peribacteroid membranes (26 and 28 kD, Z. Hong and D.P.S. Verma, unpublished data) have been shown to bind GTPyS. In a preliminary study on rice, it was shown that the binding of GTPyS to vesicles in vitro was increased by the growth hormone IAA, suggesting the role of GTPbinding proteins in cell elongation stimulated by auxin (Zaina et al., 1990). It was reported that a substantial amount of a 28-kD GTP-binding protein was translocated from the ER and Gol@fractions to the plasma membrane and chloroplasts when cells of the green alga Dunaliella salina were subjected to hypoosmotic swelling (Memon et al., 1993). A variety of approaches have been taken for the isolation of a number of cDNAs encoding small GTP-binding proteins from plants. Many of these cDNAs were isolated by using degenerate oligonucleotides corresponding to one of the consensus sequences in the GTPase superfamily. Complementation of yeast mutants with a plant cDNA library was successful in isolating a SARZ homolog cDNA from Arabidopsis (dEnfert et al., 1992). A subtraction screening strategy has been used in cloning a rabll homolog cDNA ( r g p l ) from rice (Sano and Youssefian, 1991). Low-stringency screening using heterologous probes has allowed isolation of homologous cDNAs like Rab7 from Vigna aconitifolia (Cheon et al., 1993) and rgp2 from rice (Youssefian et al., 1993). By sequencing 130 randomly selected clones from a maize leaf cDNA library, a Rab5 homolog clone was identified (Keith et al., 1993). From Arabidopsis thaliana, at least seven different clones encoding small GTP-binding proteins have been isolated (Matsui et al., 1989; Anai et al., 1991; Anuntalabhochai et al., 1991; Bednarek et al., 1994). Although their specific functions are not known, Rhal, one of the seven Arabidopsis clones, was highly homologous to Rab5, which is localized Downloaded from www.plantphysiol.org on June 7, 2015 - Published by www.plant.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. zyxwvu zyxwvutsrqponmlkj Plant Physiol. Vol. '106, 1994 Verma et al. 4 GTP CIlP cells having a11 the consensus sequences and the C -terminal Cys motif (Palmeet al., 1992). We have isolated cDNA clones encoding Rabl and Rab7 homologs from soybean and V. uconitifolia root nodules (Cheon et al., 1993). Furctionally, the plant Rablp is able to complement the yeast y p t l mutant. The expression of rab7 is enhanced significantly during nodulation, with the level of rub7 mRNA being 12 times higher than that in root meristem and leaves. This coincides with the membrane proliferation and endocytosis of Rhizobium in root nodules. Reducing the expression of these pxoteins by antisense cDNAs under the control of nodule-specific (leghemoglobin) promoter drastically affected the biol;enesis of symbiotic organelle and nodule development (Chcbon et al., 1993). Considering the extensive membrane trafficking that occurs in infected cells of root nodules and the iniportance of proper membrane biogenesis in symbiotic interaction, the root nodules provide an excellent system with which to study the basic machinery involved in vesicle-mediated transport in plants. 'Temporal and spatial control of expressio,i of these genes using tissue-specific promoters and antisense or negative complementation approaches may allow dis:,ection of the roles of these proteins in the vesicular transpor1 system. zyxwvutsrqpon "Off" f Pi Figure 1. Small GTP-binding proteins as molecular switches. The switch is turned on when a small GTP-binding protein (SMC) binds to GTP with the help of GEF. GAP promotes CTP hydrolysis, which turns the switch off. zyxwvuts zyxwvutsrqp to early endosomes. Its expression, revealed by rhal promoter-driven 0-glucuronidase activity, was found mainly in the guard cells and also in the stipules, root tips, and young leaves, and in the receptacles of flowering Arabidopsis plants (Terryn et al., 1993). It was hypothesized that Rhal may be involved in cell-plate formation or cell-wall thickening, which requires vesicle-mediated processes. A,t.RAB6, one of the Arabidopsis genes, encodes a Rab6 homolog that is localized to media1 and trans-Golgi (Goud et al., 1990; Antony et al., 1992). It complemented a yeast ypt6 mutant (Bednarek et al., 1994), demonstrating its functional conservation. However, dominant expression of a mutated Rab6p failed to inhibit vesicular transport through mammalian Golgi (Tisdale et al., 1992). Furthermore, Ypt6 is not essential for cell viability. Overexpression of mutated A.t.RAB6 gene or antisense regulation by the wild-type gene may reveal interesting information conceming the soluble protein sorting process at the trans-Gol@network. By differential screening of cDNA libraries made from wild-type and dwarfing plants induced by azaC, a DNA methylation inhibitor, Sano and Youssefian (1991) isolated a r a b l l homolog cDNA ( r g p l ) from rice. Expression of this gene is reduced in azaC-treated rice dwarfing plants. Interestingly, the expression level of a second r a b l l homolog (rgp2, which has 53% amino acid identity with r g p l ) is not affected by azaC treatment (Youssefian et al., 1993). Expression of sense and antisense rgpl in transgenic tobacco showed reduction in apical dominance and increased tillering (Kamada et al., 1992). Two rab homologs, Np-ypt3 (a r a b l l homolog) and Nt-rab5 (a rab5 homolog), have been identified in tobacco (Dallmann et al., 1992). The expression pattems of these genes are similar; expression is highest in flowers and undetectable in leaves. SARZ homolog cDNAs have been recently identified from Arabidopsis, tomato, and soybean (dEnfert et al., 1992; Davies, 1994; Z. Hong and D.P.S. Verma, unpublished data). Although the tomato SARl gene is expressed in a11 tissues tested (Davies, 1994), expression pattems of four soybean SARl genes are very different, one being preferentially expressed during nodule organogenesis (Z. Hong and D.P.S. Verma, unpublished data). Rhol, a gene implicated in the control of microfilament organization, and a rab7 homolog cDNA have also been isolated recently from pea (Drew et al., 1993; Yang and Watson, 1993). A Yptl/Rabl homolog was identified in maize coleoptile Donor Mlembrane Target Membrane Cytoplasm I + I GDP (Jqq GEF Vesicle ] I-SNARE V-SNARE SNAP SNAP NS F I I Vesicle (V-SNAI Figure 2. The SNARE hypothesis for vesicle fusion with the target membrane. According to this hypothesis, t h e V-SNAF!E protein located on the vesicles interacts with the t-SNARE found on the target membrane in t h e presence of NSF, SNAPs, small C1-P-binding proteins, and other uncharacterized components. This interaction leads to the eventual fusion of the transport vesicles with the target membrane. In step 1, the small GTP-binding protein Rab i'; recruited to the vesicle in the presence of CEF and GTP. The rwognition and interaction between V-SNARE and t-SNARE mediated by NSF and SNAP bring the vesicle to the target membrane (step 2). Hydrolysis of GTP by Rab with the help of CAP triggers the fusion of two distinct lipid bilayers (step 3). The GDP-bourid Rab is released by CDI from the membrane to the cytosol for recycling (step 4). Fusion of the vesicle to t h e target membrane also releases the cargo of the vesicle for secretion into or outside of the membrane compartments (shaded arrow). Solid arrow indicates the formation of vesicles. zyxwvut Downloaded from www.plantphysiol.org on June 7, 2015 - Published by www.plant.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. zyx zy zyxwv zyxwvu Small GTP-Binding Proteins and Membrane Biogenesis in Plants More plant genes coding for small GTP-binding proteins will continue to be isolated by means of PCR, heterologous screening, and complementation of yeast mutations. It is a much more challenging task to figure out the function of each gene product in membrane biogenesis. Although direct complementation of yeast mutants will continue to prove useful for testing the biological functions of some plant genes, many plant small GTP-binding proteins may not have counterparts in yeast or their respective mutants may not have been isolated. Although many small GTP-binding proteins have been localized on different subcellular compartments in mammalian cells (Table I), there is a conspicuous lack of information about their subcellular distribution in plant cells. Even if the basic machinery for membrane biosynthesis is conserved between mammalian and plant cells, it remains to be answered by plant cell biologists what are the small GTPbinding proteins that are responsible for biogenesis of the membrane compartments unique to plant cells, e.g. vacuoles and the peribacteroid membranes in root nodules (Fig. 3). It is apparent that these additional subcellular compartments would require specific SNAREs to be recognized as distinct target membranes. The fusion of these vesicles with respective membranes also facilitates unloading the 'cargo" through the exocytic (secretory) pathway. Such cargo in the case of plasma membrane constitutes extracellular proteins, soluble vacuolar proteins for vacuoles, and PBF proteins secreted by fusion of the vesicles with PBM. Although the sorting of some of the secretory proteins to vacuolar and plasma membrane vesicles has been worked out (see Bednarek and Raikhel, 1992; Chrispeels and Raikhel, 1992), the targeting of PBF proteins is not clear. Moreover, the mechanism of targeting of the PBM proteins seems to vary (cf. nodulin-26 versus nodulin-24; Mia0 et al., 1992; Cheon et al., 1994). This may be due to the fact that PBM is a mosaic membrane having properties common to both plasma membrane and vacuoles. Therefore, involvement of various GTP-binding 5 proteins in PBM biogenesis may be more complex. Membrane biogenesis of the organelles such as plastids, mitochondria, and peroxisomes follows different routes. Overexpression of a sense, antisense, or dominant-mutated form of a specific gene in transgenic plants should provide some clues about the role of small GTP-binding proteins in membrane biogenesis, as have been used in studies of rgpl in tobacco (Kamada et al., 1992) and Rabl and Rab7 in soybean and Vigna (Cheon et al., 1993). Eventually, development of an in vitro vesicle fusion system in plants, as has been established from yeast and mammalian cells, although difficult due to the presence of cell walls and the abundance of proteases in the central vacuole, will be essential to decipher the detailed reactions involved in specific steps of vesicle traffic and membrane biogenesis. Received April 18, 1994; accepted May 31, 1994. Copyright Clearance Center: 0032-0889/94/106/0001/06. LITERATURE CITED Anai T, Hasegawa K, Watanabe Y, Uchimiya H, Ishizaki R, Matsui M (1991) Isolation and analysis of cDNAs encoding small GTPbinding proteins of Arabidopsis thaliana. 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