Transport proteins in the plasma membrane and the secretory system

reviews 16 Yaguchi, M. et al. (1993) Amino acid sequence and spectroscopic studies of Dutch elm disease toxin, cerato-ulmin, in Dutch Elm Disease Research, Cellular and Molecular Approaches (Sticklen, M.B. and Sherald, J.L., eds), pp. 152-170, Springer Verlag 17 Bowden, C.G. et al. (1993) Isolation and characterization of the ceratoulmin toxin gene of the Dutch elm disease pathogen, Ophiostoma ulmi, Curr. Genet. 25, 323-329 18 Wessels, J.G.H. (1994) Developmental regulation of fungal cell wall formation, Annu. Rev. Phytopathol. 32, 413-437 19 Zhang, L. et al. (1994) Virus-associated down-regulation of the gene encoding cryparin, an abundant cell-surface protein from the chestnut blight fungus Cryphonectria parasitica, Gene 139, 59-64 20 Bell-Pedersen, D., Dunlap, J.C. and Lores, J.J. (1992) The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer, Genes Dev. 6, 2382-2394 21 Lauter, F-R., Russo, V.E.A. and Yanovsky, C. (1992) Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora, Genes Dev. 6, 2373-2381 22 Thau, N. et al. (1994) rodletless mutants of Aspergillus fumigatus, Inf. Immun. 62, 4380-4388 23 Parta, M. et al. (1994) HYP1, a hydrophobin gene from AspergiUus fumigatus complements the rodletless phenotype in Aspergillus nidulans, Inf. Immun. 62, 4389-4395 24 AsgeirsdSttir, S.A., van Wetter, M.A. and Wessels, J.G.H. (1995) Differential expression of genes under control of the mating-type genes in the secondary mycelium of Schizophyllym commune Microbiology 141, 1281-1288 25 Wessels, J.G.H. et al. (1995) Genetic regulation of emergent growth in Schizophyllum commune, Can. J. Bet. 73 (Suppl. 1), $273-$281

26 Stringer, M.A. and Timberlake, W.E. (1995) dewA encodes a fungal hydrophohin component of the Aspergillus spore wall, Mol. Microbiol. 16, 33-44 27 Timberlake, W.E. (1993) Translational triggering and feedback fixation in the control of funga] development, Plant Cell 5, 1453-1460 28 Cole, G.T. and Hoch, H.C., eds (1991) The Fungal Spore and Disease Initiation in Plants and Animals, Plenum Press 29 Talbot, N.J. (1995) Having a blast: exploring the pathogenicity of Magnaporthe grisea, Trends Microbiol. 3, 9-16 30 St Leger, R.J., Staples, R.C. and Roberts, D.W. (1992) Cloning and regulatory analysis of starvation stress gene, ssgA, encoding a hydrophobin-like protein from the entomopathogenic fungus, Metarhizium anisepliae, Gene 120, 119-124 31 Talbot, N.J., Ebbole, D.J. and Hamer, J.E. (1993) Identification and characterization ofMPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea, Plant Cell 5, 1575-1590 32 Martin, F. et al. Fungat gene expression during ectomycorrhiza formation, Can. J. Bet. (in press) 33 Honneger, R. (1991) Functional aspects of the lichen symbiosis, Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 553-578 34 Wessels, J.G.H. (1994) Development of fruit bodies in homobasidiomycetes, in The Mycota (Esser, K. and Lemke, P.A., eds), Vol. I, Growth, Differentiation and Sexuality (Wessels, J.G.H. and Meinhardt, F., eds), pp. 351-366, Springer-Verlag

Joseph Wessels is at the Dept of Plant Biology, University of Groningen, Kerklaan 30, 9751 NN haren, The Netherlands (j.g:h.wessels @biol:rug.nl).

Transport proteins in the plasma membrane and the secretory system o,oooc. oo,, ,ov.Raikhet The organelles of the plant secretory system and the plasma membrane each contain a specific complement of resident integral membrane proteins, which provide each organelle with some of its unique characteristics. Over the past few years, genes encoding some of these proteins have been isolated and this has allowed the function and localization of the encoded proteins to be determined. A number of the genes encode proteins involved in transport processes, both in the trafficking of proteins between membranes and the transport of solutes across membranes. However, little is known about the majority of membrane proteins within the endomembrane system. A more complete understanding of the processes occurring within this system awaits the identification and analysis of many more of its components and the interactions between them. This will also allow important questions to be addressed regarding the mechanisms by which membrane proteins are correctly localized and assembled in the secretory pathway.

he plant endomembrane system, or secretory pathway, consists of a series of organelles that include the endoplasmic reticulum (ER), Golgi apparatus and vacuole, as well as transport vesicles connecting these compartments and the plasma membrane (Fig. 1). Transport vesicles carry proteins, lipids and polysaccharides between the organelles, and to the cell's exterior, in a series of budding and fusion

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events. Each organelle of the secretory pathway contains a specific complement of soluble and membrane proteins, which determine some of the unique properties of the organelle: sorting signals are required for the targeting and retention of proteins in most of these compartments. In recent.years, a number of proteins have been identifled that reside in membranes of the plant secretory system January 1996, Vol. 1, No. 1

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Intermediate f ER compartment cis

Plasma membrane

Golgi medial

trans

TGN

membrane topology will now allow iraportant questions to be addressed regarding their transport and assembly.

Proteins involved in vesicular trafficking Trafficking between the ER and Golgi apparatus Several cDNA clones have now been isolated encoding membrane proteins that are themselves involved in the I transport and sorting of proteins in the plant secretory pathway. Sec12p is an integral membrane glycoprotein of the yeast ER that is required for the formation of transport vesicles. Arabidopsis cDNA clones were also isolated, ~_J ~ O . (..Secretion which show homology to SEC12 and are able to complement the yeast sec12 mutation, suggesting that they function in the same way 1. Resident soluble proteins of the Fig. 1. The secretory system and the plasma membrane. Anterograde transport ER contain a C-terminal tetrapeptide through this system begins at the endoplasmic reticulum (ER) and occurs by the motif that mediates their localization budding of vesicles from one organelle and their fusion with the next. At the transin the organelle. As some of these proGolgi network (TGN), vesicles are targeted to either the plasma membrane for reins are modified by enzymes found in secretion of vesicle contents or to the vacuole. the Golgi apparatus, it has been suggested that they can escape from the ER and are retrieved by a specific sortand perform a variety of functions. Some of these proteins ing mechanism. A receptor protein (Erd2p) was identified are involved in the processes of vesicular trafficking; many in yeast that is found at the cis-Golgi, and may bind to others function in the transport of small molecules across and retrieve ER-resident proteins back to the ER. An membranes. The mechanisms by which plant membrane Arabidopsis homolog of the ERD2 gene has been cloned proteins are transported to, and retained in, the appropriate (called aERD2) that encodes a highly hydrophobic protein organelle are still obscure. The cloning of genes encoding with seven putative transmembrane domains. The aERD2 these proteins and the prediction of their structure and gene is able to complement a yeast erd2 mutant, indicating that the yeast and Arabidopsis proteins have similar functions and that aErd2p may also be located at the cis-Gol~. The expression of aERD2, and other genes encoding proteins at this stage of the secretory pathway, was investigated by studying transcript levels under various developmental and environmental conditions2. Stress conditions affecting the secretory pathway (tunicamycin treatment or cold shock) cause an increase in expression of aERD2. This suggests - Syntaxin/t-SNARE • that the secretory pathway can be regu- { ~ VAMP/v-SNARE lated in response to environmental conTarget (accepter) membrane ditions. Varying levels of expression of E:d NSF the ERD2 gene are observed in different ~ a-SNAP tissue and cell types. The highest levels @ SNAP-25 ofERD2 mRNA are found in roots. Very low transcript levels are observed in Fig. 2. General model for the docking of a vesicle with its target membrane [the leaves throughout development, but with SNARE (SNAP-receptor) hypothesis]. Proteins at the target membrane, such as an increased accumulation of transcript syntaxin and SNAP-25 (a synaptosomal-associated protein of 25 kDa) interact in trichomes in older leaves. This could with a protein in the vesicle membrane, VAMP (vesicle-associated membrane protein), along with the soluble proteins NSF (N-ethylmaleimide sensitive factor) and reflect differing levels of secretory patha-SNAP (soluble NSF attachment protein), to form a docking complex. Different way activity in roots and leaves. syntaxin and VAMP isoforms have been found on various organelles in maremalian and yeast cells, and this has been proposed to provide specificity for the Transport to the vacuole fusion of a vesicle with only the correct target organelle. The transport of proteins to the vacuole has been widely studied in plants,

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Janua~1996,Vol.1, No. 1

reviews and soluble proteins have been shown to require a targeting signal for deposition in the vacuole. Three types of signals have been identified: C-terminal propeptides (CTPPs), Nterminal propeptides (NTPPs), and regions of the mature protein 3. By analogy with other systems, it is expected that receptors, which mediate vacuolar sorting, exist for these signals. Recently, a potential receptor was identified and purified from clathrin-coated vesicles, which transport vacuolar proteins from the trans-Golgi network to the vacuole. This 80 kDa integral membrane protein binds to an affinity column consisting of the pro-aleurain NTPP. The protein is also able to bind to peptides representing the NTPP of sweet potato (Ipomoea batata) sporamin and the C-terminal targeting sequence of Brazil nut (Bertholletia excelsa) 2S-albumin, but not to the CTPP of barley lectin 4. This indicates that different receptors may exist for subsets of vacuolar proteins. However, there are no similarities in the primary structures of the NTPPs and the C-terminal sequence of 2S albumin. PEP12 is a yeast gene encoding a protein belonging to the syntaxin family. Syntaxins are integral membrane proteins, thought to act as receptors for transport vesicles arriving at and fusing with the membrane, with the bulk of the protein facing the cytosol. Upon docking of a vesicle with its target membrane, syntaxin interacts with a number of proteins, both in the vesicle membrane and in the cytosol to form a docking complex (Fig. 2). Different isoforms of syntaxin have been proposed to reside on different cellular membranes and to provide specificity for the docking and fusion reaction. A yeast pep12 mutant is defective in the targeting of proteins to the vacuole, and the Pep12 protein is thus likely to function in vesicle transport between the trans-Golgi network and the vacuole. An Arabidopsis cDNA (aPEP12) was isolated by functional complementation of the yeastpep12 mutant and found to be homologous to the yeast PEP12 gene and other members of the syntaxin family 5. The aPep12 protein is therefore a potential component of the plant vacuolar transport machinery. Transport of solutes across membranes Many of the membrane proteins which have been identifled within the secretory pathway function in transporting small molecules across membranes. For some of these proreins, features such as their structure, topology and location within the pathway have now been determined, although it is still unclear how assembly into the correct membrane is achieved and which cellular factors mediate the process. Plasma membrane H÷-ATPase

In plants, the primary active transport system at the plasma membrane is the proton translocating ATPase (H÷ATPase), which couples ATP hydrolysis to the transport of protons from the cytosol across the plasma membrane. This produces a membrane potential, consisting of an electrical potential (negative inside) and a pH gradient (more acidic outside) that can be used to drive the uptake of solutes into the cell~. The H÷-ATPase was the first plant plasma-mere-. brane protein to be purified to homogeneity. It is a 100 kDa protein that forms a phosphorylated intermediate during each cycle of ATP hydrolysis. This has led to its classification as a P-type (plasma membrane) ATPase; tryptic peptides from the purified protein also show homology to P-type ATPases from other organisms 7. Genomic and cDNA clones encoding H÷-ATPases have been isolated from a number of species, with the most exten-

sive analysis being performed on Arabidopsis thaliana. Surprisingly, a large family of genes (AHAs, for Arabidopsis H+-ATPase) encoding H÷-ATPases have been identified, and differential regulation of mRNA expression for these genes has been demonstrated inArabidopsis and tobacco7,8. Clones for the H÷-ATPase from both of these plants have been found to contain a short open-reading-frame in the 5' region upstream of the main reading frame, which may be involved in translational gene-regulation. Developmental and growth conditions have also been seen to regulate H÷-ATPase expression 9, as has cytosolic calcium concentration ~°. The expression of some plant genes in yeast demonstrated that they do encode functional H÷-ATPases with properties similar to the yeast plasma membrane H÷-ATPase. A comparison between the properties of AHA1, AHA2 and AHA3, expressed in yeast, has shown that these three isoforms display different pH optima, KmSfor ATP and inhibitor sensitivities, suggesting that there are functional differences between the isoforms 7. To determine the cell-specific locations of expression of the different AHA genes, fusions were made between their 5' upstream regions and the reporter gene ~-glucuronidase (GUS)~. The analysis of GUS activity by light microscopy indicated the expression pattern of the AHA genes. For example, AHAIO was shown to be expressed primarily in developing seeds8. In addition, detailed mRNA analysis indicated that AHA9 was expressed only in anther tissues 11. It has been hypothesized that each AHA-protein isoform is expressed in a particular transport tissue and thus could create the driving force for solute transport in that tissue. This has been demonstrated recently for the AHA3 protein, which was found by immunocytochemical localization to be restricted to the plasma membrane of phloem companion cells and, therefore, may be involved in phloem loading 12.

VacuolarH÷-ATPase Internal membranes of the plant secretory pathway contain vacuolar (V-type) H+-ATPases. These enzymes generate a proton-motive force that drives solute transport across the membrane, as discussed for the P-type H+-ATPases. The V-ATPases (V-type H+-ATPases) are multimeric enzymes consisting of two complexes, one of which is the membranespanning Vo complex, and the other is the catalytic V1 complex, found on the cytosolic face of the membrane. Electronmicroscope studies have shown that the V1 complex forms a 'ball and stalk' structure similar to that of the F-type H÷-ATPases of mitochondria and chloroplasts. Three major subunits of the V-ATPase have been identified, all of which have several isoforms, and cDNA clones have been isolated that encode these proteins. The V1 complex contains a 70 kDa catalytic subunit and a 60 kDa regulatory subunit, both of which contain nucleotide binding sites. The Vo complex contains six copies of a 16 kDa proteolipid subunit that is a hydrophobic protein with four putative membrane-spanning domains. The V-ATPase also contains several minor subunits; purified V-ATPase from oat roots has been shown to consist of ten different polypeptides. Six of these polypeptides were found in a large peripheral complex that could be released from the membrane by chaotropic anions. This release led to loss of ATPase and H÷-pumping activities, which were restored upon reassembly of the complex following dialysis. The subunit composition of V-ATPases purified from different plant species varies, which could reflect developmental or tissue-specific Janua~ 1996, Vokl, No. 1

17

reviews regulation of expression of subunit isoforms, as well as plasma membrane and one at the tonoplast of tomato differences between species 13. roots21. This raises the interesting question of how the two The purified oat V-ATPase is active in both ATPase func- different isoforms are targeted to discrete subcellular tion and H÷-translocation, when reconstituted into proteo- locations. liposomes, demonstrating that the known components are sufficient for activity. This activity is stimulated by chlo- K + transport ride, implicating cytoplasmic chloride concentrations in the One major nutrient whose uptake is indirectly coupled to regulation of the V-ATPase in vivo 14. The ratio of H ÷ ions the activity of the P-type H+-ATPase is potassium (K), which translocated per ATP hydrolyzed has been determined for is required for plant growth and osmoregulation. At least the red beet (Beta vulgaris) V-ATPase, and shown to be two transport systems for K÷ uptake exist in the plasma dependent on the cytoplasmic and vacuolar pH, indicating membrane: a high affinity and a low affinity system. that these pHs may also regulate the enzyme activity1S. Two plasma membrane K+ channels have been cloned by Whereas V-ATPases are not usually phosphorylated during functional complementation of yeast mutants defective in the reaction, the V-ATPase found at the tonoplast of Acer K+ uptake, an approach that has proved powerful for the pseudoplatanus has been suggested to operate via a phos- isolation of cDNAs encoding plant membrane proteins. phoenzyme intermediate, in a similar manner to the These mutants require millimolar concentrations of K÷ for P-type H+-ATPases1~. growth, whereas wild-type yeast can grow on media conThe assembly of the V-ATPase of oat roots has been stud- taining only micromo]ar concentrations. The mutated genes ied by Herman and co-workers ~. In mature cells, the major encode the putative K+-transporters TRK1, a high affinity subunits of the V1 complex are associated with vacuolar transporter, and TRK2, a low affinity transporter. membranes. In immature cells, these subunits are mainly Arabidopsis genes were identified that restored the ability found at the ER. It has been suggested, therefore, that the of the mutant yeast to grow on low K+ concentrations, and V1complex assembles with the Vo complex on the ER, rather were named KAT1 and AKT1. These genes share no homthan directly at the vacuole. However, the mechanism of ology with the yeast TRK genes, but an intriguing similarity this assembly is not known: for example, are molecular has been observed to the shaker genes, which encode K+ chaperones involved in the process, or does self-assembly channel proteins in Drosophila and mammals. The exoccur? pression of the KAT1 gene in Xenopus oocytes followed by patch-clamp analysis confirmed that the encoded protein is Vacuolar H+-pyrophosphatase responsible for K+ uptake 7. Recently, a cDNA has been isoThe plant vacuolar membrane also contains another elec- lated from Arabidopsis encoding a protein homologous to trogenic proton pump, the H+-translocating pyrophos- the ~ subunit of mammalian K+ channels, a 39 kDa hydrophatase (H+-PPase). Inorganic pyrophosphate is used as philic protein that may associate with the channel proteins the energy source for the translocation of protons across and act in a regulatory role22. The function of this ~ subunit the membrane, and H + translocation is dependent on the homolog remains unclear. presence of potassium ions (K+/in the cytosol. This enzyme While low-affinity K÷ uptake occurs via channels, highis unusual in that it seems to be restricted to plant cells affinity uptake, required when the external K+ concenand some photosynthetic bacteria is. In contrast to the tration is low, appears to be mediated by a thermodynamiH÷-ATPase, the H÷-PPase appears to consist of a single cally active process. The approach of complementation of a (approximately 80kDa) subunit; the expression, in yeast, of yeast mutant similarly allowed the isolation of a cDNA from this subunit from Arabidopsis demonstrated that it is suffi- wheat that enables the yeast mutant to take up K÷(Ref. 23). cient for all known catalytic functions of the enzyme19. The The encoded protein (HKT1) shows a weak homology to K+ analysis of H+-PPase amino acid sequenceslS,2° indicates uptake transporters from yeast and is a K+-H+ co-transthat the protein is very hydrophobic, with 13 to 16 amphi- porter. The HKT1 gene is expressed in the root cortex and pathic membrane-spanning regions. A putative catalytic cell layers surrounding the leaf vascular tissue, as would be motif, observed in soluble PPases, is also found in a cytosolic predicted for a nutrient uptake system. Surprisingly, while loop of the H+-PPase. The role in vivo of the H+-PPase is electrophysiological data indicate the presence of outwardunclear. It has been suggested that the enzyme may func- rectifying K+channel in plant cells, none has yet been cloned. tion in translocation of both H + and K÷ ions across the tonoplast, allowing K+ transport against the membrane po- Transport of nitrogenous compounds tential. Alternatively, it may act to scavenge the free energy Several plant genes encoding transporters for nitrogenof pyrophosphate, rather than allowing this energy to ous compounds have now been identified using a variety be lost 18. of methods. A nitrate-inducible nitrate transporter gene (CHL1) from Arabidopsis was cloned using a transferredCa2+transport DNA (T-DNA) tagged mutant that was resistant to chlorate; A cDNA (designated LCA) has been isolated from tomato chlorate is a nitrate analogue, reduced in the plant to chlowhich shows high homology to ER calcium-dependent rite, which is toxic. The protein was expressed in Xenopus ATPases from other organisms. These are P-type ATPases oocytes, demonstrating that it encodes a functional electrowhich transport two calcium ions (Ca 2+) per ATP genic nitrate-H + co-transporter, presumably found at the hydrolyzed. The mRNA level has been shown to increase in plasma membrane of plant cells 24. response to salt stress: this may be caused by a disruption A high-affinity ammonium transporter and amino acid of intracellular Ca2+ concentration by high salt conditions. transporters have been identified in Arabidopsis, also by Interestingly, immunolocalization of the protein encoded complementation of yeast mutants deficient in the respective by the LCA gene, and Ca2+-ATPase activity assays, suggest transporters 25. Multiple amino acid transporters appear to that LCA may encode two Ca2+-ATPase isoforms, one at the exist, with differing substrate specificities and mechanisms. 18

January 1996, Vol. 1, No. 1

reviews Sugar transport Molecular data are available for a number of sugar transporters thought to be involved in the distribution of sugars between different plant tissues26,27. The first H+-coupled cotransporter to be cloned from a eukaryote was the Chlorella hexose transporter. The protein contains 12 putative membrane-spanning domains and is a member of a superfamily of transporters found in many different organisms. The first higher plant glucose transporter was cloned from Arabidopsis by using a cDNA from Chlorella as a probe, and the function of the protein has been determined by expression in yeast. The yeast complementation approach was used to isolate the first cDNA encoding a sucrose carrier (from spinach). Genes encoding sugar transporters from other plant species have now also been identified ~6. Interestingly, a putative sugar transporter from sugar beet has been cloned recently that is localized in the tonoplast, and may thus be involved in the partitioning of sugar between the vacuole and cytosol~8.

Major intrinsic protein family A family of proteins that has received much attention recently in plants is the aquaporins, which mediate the passage of water across membranes. They are part of a larger family of related proteins, the major intrinsic protein (MIP) family, which all appear to consist of six membrane-spanning domains with their amino- and carboxy-termini facing the cytosol. Some of the MIPs have been shown to exist as tetramers, although the monomer appears to be the functional unit. Whereas the aqnaporins transport water, other members of the MIP family transport solutes such as ions and glycerol 29,3o.In plants, a number of MIP homologs have been identified in both the plasma membrane and tonoplast. Tonoplast intrinsic protein (TIP) is a member of this family from bean (Phaseolus vulgaris), and different isoforms are present in different tissues 3o. The function of ~-TIP has been determined by expression of the protein in oocytes2~. When oocytes expressing ,/-TIP are moved to a hypotonic solution, they swell and burst rapidly, when compared with control oocytes. The presence of ~/-TIP in the oocyte membrane thus increases their permeability to water by approximately five- to tenfold. Many other plant genes have been isolated that encode members of the MIP family, including plasma membrane aquaporins 31,32and nodulin-26, a phosphorylated protein found in the peribacteroid merebrane of root nodules in soybean33. Cell and developmental specific expression patterns have been observed for various aquaporins, and certain physiological conditions can induce aquaporin expression, for example, water deprivation29, 34. The function of a MIP homolog in plants was addressed by expressing an antisense construct of the blue light inducible AthH2 gene ofArabidopsis ~4. Leafprotoplasts from the antisense plants show reduced water uptake when compared with control plants. Recently, the seed-specific aquaporin a-TIP, which has been suggested to increase the transport of water across the tonoplast of protein storage vacuoles, was shown to be reguiated by phosphorylation 35. The a-TIP protein has three potential phosphorylation sites, and mutation of these sites reduces its ability to transport water in Xenopus oocytes. Cyclic AMP-dependent protein kinase A (PKA) is able to phosphorylate a-TIP, and stimulation of the endogenous

PKA activity in oocytes by cAMP agonists increased the water transport activity of ~-TIP. Phosphorylation by a specific protein kinase may thus provide a means for the regulation of ~-TIP activity in the seed during the early stages of germination. In plants, a MIP gene family therefore exists with different isoforms found in various cell membranes and tissues, and the diversity of this family may reflect differing channel requirements dependent on the proteins' location and expression pattern. Targeting signals must exist within the isoforms to ensure their correct subcellular localization, although these signals remain largely obscure (see below).

Membrane protein targeting signals To assume their proper function in the cell, membrane proteins must be correctly targeted and transported to their site of action. While targeting signals of several soluble proteins of the plant secretory pathway have been defined, there is little information available on the targeting signals within membrane proteins 3. In particular, it is not known whether the 'default' compartment for secretory membrane proteins is the plasma membrane or the tonoplast, whereas secretion is known to be the 'default' pathway for soluble proteins. The targeting of a-TIP to the tonoplast has been studied by creating a fusion protein consisting of a portion of a-TIP fused to the reporter protein, phosphinotricine acetyltransferase 36. This demonstrated that a segment of a-TIP, containing the sixth transmembrane domain and the C-terminal cytoplasmic tail, is sufficient for vacuolar localization. A deletion mutant of a-TIP, lacking the cytoplasmic tail, is still found at the tonoplast, which implies that the transmembrane domain alone is sufficient for vacuolar targeting. In addition, a-TIP has been shown to be transported to the vacuole by a different mechanism to soluble vacuolar proteins 37. However, the question of the location of the 'default' compartment for membrane proteins in the plant secretory pathway remains to be answered. Conclusions The study of plant membrane proteins that reside in the various organelles of the secretory pathway has been until recently hampered by the difficulties associated with purifying these proteins, and cloning their genes by conventiona] means. Genomic or cDNA clones are now available for a number of proteins, and it is likely that more will be isolated in the near future. In particular, the complementation of existing yeast mutants with plant cDNAs has proved in some cases to be a fruitful method for identifying functional plant homologs of yeast proteins, when the sequence identity between the genes may be too low for the screening of libraries using a heterologous probe. However, this does not circumvent the problem of the isolation of genes encoding plant-specific membrane proteins, which have no homologs in other systems. These are perhaps some of the most interesting and difficult problems to address, and imaginative approaches will be required to isolate genes and elucidate the functions of the encoded proteins. The Golgi apparatus in plant cells has some unique functions, by which many of the components of the cell wall are synthesized, and thus will contain many interesting and novel proteins. However, very little is known about this compartment at the molecular level. The isolation of genes is, of course, only a first step to understanding the function

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reviews and regulation of the proteins in vivo. Epitope tagging and promoter fusions to reporter genes are beginning to provide indications about the localization and expression pattern of the gene products. Using antisense expression, in combination with dominant negative-mutations, we can begin to elucidate the function of pro-teins in the context of the whole plant. The availability of genes encoding homologous proteins located in various compartments will also allow the study of targeting signals and mechanisms by domainswapping experiments, the creation of deletion mutants and gene fusions, and the determination of the proteins' intracellular location. Acknowledgements

Support was provided by the National Science Foundation and the US Department of Energy to N.V.R. References 1 d'Enfert, C., Gensse, M. and Gaillardin, C. (1992) Fission yeast and a plant have functional homologues of the Sarl and See12 proteins involved in ER to Golgi traffic in budding yeast, EMBO J. 11, 4205-4211 2 Bar-Peled, M. et al. (1995) Expression and regulation ofaERD2, a gene encoding the KDEL receptor homolog in plants, and other genes encoding proteins involved in ER-Golgi vesicular trafficking, Plant Ceil 7, 667-676 3 Gal, S. and Raikhel, N.V. (1993) Protein sorting in the endomembrane system of plant cells, Curr. Opin. Ceil Biol. 5, 636-640 4 Kirsch, T. et al. (1995) Specificity of a potential vacuolar targeting receptor for vacuolar targeting information, Mol. Biol. Cell 16, 104a 5 Bassham, D.C. et al. (1995) An Arabidopsis syntaxin homologue isolated by functional complementation of a yeast pep12 mutant, Proc. Natl Acad. Sci. USA 92, 7262-7266 6 Serrano, R. (1989) Structure and function of plasma membrane ATPase, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 61-94 7 Sussman, M.R. (1994) Molecular analysis of proteins in the plant plasma membrane, Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 211-234 8 Harper, J.F., Manney, L. and Sussman, M.R. (1994) The plasma membrane H+-ATPase gene family in Arabidopsis: genomic sequence of AHAIO which is expressed primarily in developing seeds, Mol. Gen. Genet. 244, 572-587 9 Michelet, B. et al. (1994) A plant plasma membrane proton-ATPase gene is regulated by development and environment and shows signs of a translational regulation, Plant Cell 6, 1375-1389 10 Kinoshita, T., Nishimura, M. and Shimazaki, K. (1995) Cytesolic concentration of Ca2÷regulates the plasma membrane H+-ATPase in guard cells of lava bean, Plant Cell 7, 1333-1342 11 Houln~, G. and Boutry, M. (1994) Identification of an Arabidopsis thaliana gene encoding a plasma membrane H+-ATPase whose expression is restricted to anther tissues, Plant J. 5, 311-317 12 DeWitt, N.D. and Sussman, M.R. ImmunocytoIogical localization of an epitope-tagged plasma membrane proton pump (H*-ATPase) in phloem companion cells, Plant Cell (in press) 13 Sze, H., Ward, J.M. and Lai, S. (1992) Vacuolar H+-translocating ATPases from plants: structure, function and isoforms, J. Bioenerg. Biomembranes 24, 371-381 14 Ward, J.M. and Sze, H. (1992) Proton transport activity of the purified vacuolar H+-ATPase from oats, Plant Physiol. 99, 925-931 15 Davies, J.M., Hunt, I. and Sanders, D. (1994) Vacuolar H+-pumping ATPase variable transport coupling ratio controlled by pH, Proc. Natl Acad. Sci. USA 91, 8547-8551 16 Maguin, T. et al. (1995) The tonoplast H+-ATPase of Acer pseudoplatanus is a vacuolar-type ATPase that operates with a phosphoenzyme intermediate, Plant Physiol. 109,285-292 17 Herman, E.M. et al. (1994) Vacuolar-type H+-ATPases are associated with the endoplasmic reticulum and provacuoles of root tip cells, Plant Physiol. 106, 1313-1324

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Diane Bassham and Natasha Raikhel are at the DOE Plant Research Laboratory, MichiganState University, East Lansing, MI 48824-1312, USA.