Protein sorting in the endomembrane system of plant cells.

Although many properties of the targeting of plant endomembrane proteins are similar to mammalian and yeast systems, several clear differences are found that will be stressed in this review. In the past year, significant advances in our understanding of storage protein segregation in the endoplasmic reticulum, compartmentation of Golgi, and the signals for vacuolar protein targeting have been made. This work will form the basis for determining the mechanism of these sorting phenomena.

Unique features of the plant vacuolar sorting machinery.

Multiple types of vacuoles can exist within the same plant cell, and different vesicle-trafficking pathways transport proteins to each of them. Recent work has identified proteins unique to each vacuole type, and the transport pathways have begun to be elucidated. Plant trafficking proteins are usually encoded by small gene families, the different members of which have distinct functions in the endomembrane system.

VACUOLELESS1 is an essential gene required for vacuole formation and morphogenesis in Arabidopsis.

Most plant cells are characterized by the presence of a large central vacuole that in differentiated cells accounts for more than 90% of the total volume. We have undertaken a genetic screen to look for mutants that are affected in the formation of vacuoles in plants. In this study, we report that inactivation of the Arabidopsis gene VACUOLELESS1 (VCL1) blocks vacuole formation and alters the pattern of cell division orientation and cell elongation in the embryo. Consistent with a role in vacuole biogenesis, we show that VCL1 encodes the Arabidopsis ortholog of yeast Vps16p. In contrast to yeast mutants that lack a vacuolar compartment but are viable and morphologically normal, loss of the plant vacuole leads to aberrant morphogenesis and embryonic lethality.

Different sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of distinct sorting machineries in tobacco cells.

Vacuolar matrix proteins in plant cells are sorted from the secretory pathway to the vacuoles at the Golgi apparatus. Previously, we reported that the NH2-terminal propeptide (NTPP) of the sporamin precursor and the COOH-terminal propeptide (CTPP) of the barley lectin precursor contain information for vacuolar sorting. To analyze whether these propeptides are interchangeable, we expressed constructs consisting of wild-type or mutated NTPP with the mature part of barley lectin and sporamin with CTPP and mutated NTPP in tobacco BY-2 cells. The vacuolar localization of these constructs indicated that the signals were interchangeable. We next analyzed the effect of wortmannin, a specific inhibitor of mammalian phosphatidylinositol (PI) 3-kinase on vacuolar delivery by NTPP and CTPP in tobacco cells. Pulse-chase analysis indicated that 33 microM wortmannin caused almost complete inhibition of CTPP-mediated transport to the vacuoles, while NTPP-mediated transport displayed almost no sensitivity to wortmannin at this concentration. This indicates that there are at least two different mechanisms for vacuolar sorting in tobacco cells, and the CTPP-mediated pathway is sensitive to wortmannin. We compared the dose dependencies of wortmannin on the inhibition of CTPP-mediated vacuolar delivery of proteins and on the inhibition of the synthesis of phospholipids in tobacco cells. Wortmannin inhibited PI 3- and PI 4-kinase activities and phospholipid synthesis. Missorting caused by wortmannin displays a dose dependency that is similar to the dose dependency for the inhibition of synthesis of PI 4-phosphate and major phospholipids. This is different, however, than the inhibition of synthesis of PI 3-phosphate. Thus, the synthesis of phospholipids could be involved in CTPP-mediated vacuolar transport.

Immunocytochemical localization of wheat germ agglutinin in wheat.

Immunocytological techniques were developed to localize the plant lectin, wheat germ agglutinin (WGA), in the tissues and cells of wheat plants. In a previous study we demonstrated with a radioimmunoassay that the lectin is present in wheat embryos and adult plants both in the roots and at the base of the stem. We have now found, using rhodamine, peroxidase, and ferritin-labeled secondary antibodies, that WGA is located in cells and tissues that establish direct contact with the soil during germination and growth of the plant In the embryo, WGA is found in the surface layer of the radicle, the first adventitious roots, the coleoptile, and the scutellum. Although found throughout the coleorhiza and epiblast, it is at its highest levels within the cells at the surface of these organs. In adult plants, WGA is located only in the caps and tips of adventitious roots. Reaction product for WGA was not visualized in embryonic or adult leaves or in other tissues of adult plants. At the subcellular level, WGA is located at the periphery of protein bodies, within electron-translucent regions of the cytoplasm, and at the cell wall-protoplast interface. Since WGA is found at potential infection sites and is known to have fungicidal properties, it may function in the defense against fungal pathogens.

The plant vacuolar sorting receptor AtELP is involved in transport of NH(2)-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana.

Many soluble plant vacuolar proteins are sorted away from secreted proteins into small vesicles at the trans-Golgi network by transmembrane cargo receptors. Cleavable vacuolar sorting signals include the NH(2)-terminal propeptide (NTPP) present in sweet potato sporamin (Spo) and the COOH-terminal propeptide (CTPP) present in barley lectin (BL). These two proteins have been found to be transported by different mechanisms to the vacuole. We examined the ability of the vacuolar cargo receptor AtELP to interact with the sorting signals of heterologous and endogenous plant vacuolar proteins in mediating vacuolar transport in Arabidopsis thaliana. AtELP extracted from microsomes was found to interact with the NTPPs of barley aleurain and Spo, but not with the CTPPs of BL or tobacco chitinase, in a pH-dependent and sequence-specific manner. In addition, EM studies revealed the colocalization of AtELP with NTPP-Spo at the Golgi apparatus, but not with BL-CTPP in roots of transgenic Arabidopsis plants. Further, we found that AtELP interacts in a similar manner with the NTPP of the endogenous vacuolar protein AtALEU (Arabidopsis thaliana Aleu), a protein highly homologous to barley aleurain. We hypothesize that AtELP functions as a vacuolar sorting receptor involved in the targeting of NTPP-, but not CTPP-containing proteins in Arabidopsis.

Localization of wheat germ agglutinin--like lectins in various species of the gramineae.

Antigenically similar chitin-binding lectins are present in the embryos of wheat, barley, and rye, members of the Triticeae tribe of the grass family (Gramineae). However, the lectins display different localization patterns in these embryos. Lectin is absent from the coleoptile of barley but is present in the outer surface cells of this organ in wheat and in both inner and outer surface cells of rye coleoptiles. All three cereals contain lectin at the periphery of embryonic roots. Similar lectins were not detected in oats and pearl millet, members of other tribes of the Gramineae. Rice, a species only distantty related to wheat, contains a lectin that is antigenically similar to the other cereal lectins and located at the periphery of embryonic roots and throughut the coleoptile.

Xyloglucan fucosyltransferase, an enzyme involved in plant cell wall biosynthesis.

Cell walls are crucial for development, signal transduction, and disease resistance in plants. Cell walls are made of cellulose, hemicelluloses, and pectins. Xyloglucan (XG), the principal load-bearing hemicellulose of dicotyledonous plants, has a terminal fucosyl residue. A 60-kilodalton fucosyltransferase (FTase) that adds this residue was purified from pea epicotyls. Peptide sequence information from the pea FTase allowed the cloning of a homologous gene, AtFT1, from Arabidopsis. Antibodies raised against recombinant AtFTase immunoprecipitate FTase enzyme activity from solubilized Arabidopsis membrane proteins, and AtFT1 expressed in mammalian COS cells results in the presence of XG FTase activity in these cells.

Expression and Regulation of aERD2, a Gene Encoding the KDEL Receptor Homolog in Plants, and Other Genes Encoding Proteins Involved in ER-Golgi Vesicular Trafficking.

aERD2 and aSAR1 of Arabidopsis are functional homologs of yeast genes encoding proteins essential for endoplasmic reticulum (ER)-to-Golgi transport. The regulation of these secretory pathway genes in yeast, mammals, and plants is not known. High levels of expression of aERD2 and aSAR1 were observed in roots, flowers, and inflorescence stems, with the highest levels being detected in roots. The aSAR1 transcript levels were highest in young leaves and declined during leaf maturation. Low levels of aERD2 were detected in both young and fully mature leaves when compared with roots. In situ hybridization showed that trichomes accumulate more aERD2 transcript as the leaf expands, whereas aSAR1 is expressed equally in all leaf cell types. Treating plants with tunicamycin, a drug that blocks N-glycosylation in the ER, or with cold shock, known to block secretory protein transport, led to a marked accumulation of aERD2 and aSAR1 transcripts. The Arabidopsis ARF gene, which encodes a GTPase probably involved in Golgi vesicle traffic, was not affected by these treatments. This study is an essential first step toward understanding the regulation of genes that encode proteins involved in vesicular trafficking.

Interactions between syntaxins identify at least five SNARE complexes within the Golgi/prevacuolar system of the Arabidopsis cell.

The syntaxin family of soluble N-ethyl maleimide sensitive factor adaptor protein receptors (SNAREs) is known to play an important role in the fusion of transport vesicles with specific organelles. Twenty-four syntaxins are encoded in the genome of the model plant Arabidopsis thaliana. These 24 genes are found in 10 gene families and have been reclassified as syntaxins of plants (SYPs). Some of these gene families have been previously characterized, with the SYP2-type syntaxins being found in the prevacuolar compartment (PVC) and the SYP4-type syntaxins on the trans-Golgi network (TGN). Here we report on two previously uncharacterized syntaxin groups. The SYP5 group is encoded by a two-member gene family, whereas SYP61 is a single gene. Both types of syntaxins are localized to multiple compartments of the endomembrane system, including the TGN and the PVC. These two groups of syntaxins form SNARE complexes with each other, and with other Arabidopsis SNAREs. On the TGN, SYP61 forms complexes with the SNARE VTI12 and either SYP41 or SYP42. SYP51 and SYP61 interact with each other and with VTI12, most likely also on the TGN. On the PVC, a SYP5-type syntaxin interacts specifically with a SYP2-type syntaxin, as well as the SNARE VTI11, forming a SNARE complex likely involved in TGN-to-PVC trafficking.

The plant vesicle-associated SNARE AtVTI1a likely mediates vesicle transport from the trans-Golgi network to the prevacuolar compartment.

Membrane traffic in eukaryotic cells relies on recognition between v-SNAREs on transport vesicles and t-SNAREs on target membranes. Here we report the identification of AtVTI1a and AtVTI1b, two Arabidopsis homologues of the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast. AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32 and 30% identical to the yeast protein, respectively. By suppressing defects found in specific strains of yeast vti1 temperature-sensitive mutants, we show that AtVTI1a can substitute for Vti1p in Golgi-to-prevacuolar compartment (PVC) transport, whereas AtVTI1b substitutes in two alternative pathways: the vacuolar import of alkaline phosphatase and the so-called cytosol-to-vacuole pathway used by aminopeptidase I. Both AtVTI1a and AtVTI1b are expressed in all major organs of Arabidopsis. Using subcellular fractionation and immunoelectron microscopy, we show that AtVTI1a colocalizes with the putative vacuolar cargo receptor AtELP on the trans-Golgi network and the PVC. AtVTI1a also colocalizes with the t-SNARE AtPEP12p to the PVC. In addition, AtVTI1a and AtPEP12p can be coimmunoprecipitated from plant cell extracts. We propose that AtVTI1a functions as a v-SNARE responsible for targeting AtELP-containing vesicles from the trans-Golgi network to the PVC, and that AtVTI1b is involved in a different membrane transport process.

AtVPS45 complex formation at the trans-Golgi network.

The Sec1p family of proteins are thought to be involved in the regulation of vesicle fusion reactions through interaction with t-SNAREs (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) at the target membrane. AtVPS45 is a member of this family from Arabidopsis thaliana that we now demonstrate to be present on the trans-Golgi network (TGN), where it colocalizes with the vacuolar cargo receptor AtELP. Unlike yeast Vps45p, AtVPS45 does not interact with, or colocalize with, the prevacuolar t-SNARE AtPEP12. Instead, AtVPS45 interacts with two t-SNAREs, AtTLG2a and AtTLG2b, that show similarity to the yeast t-SNARE Tlg2p. AtTLG2a and -b each colocalize with AtVPS45 at the TGN; however, AtTLG2a is in a different region of the TGN than AtTLG2b by immunogold electron microscopy. Therefore, we propose that complexes containing AtVPS45 and either AtTLG2a or -b define functional subdomains of the TGN and may be required for different trafficking events. Among other Arabidopsis SNAREs, AtVPS45 antibodies preferentially coprecipitate AtVTI1b over the closely related isoform AtVTI1a, implying that AtVTI1a and AtVTI1b also have distinct functions within the cell. These data point to a functional complexity within the plant secretory pathway, where proteins encoded by gene families have specialized functions, rather than functional redundancy.

Crystallization and preliminary X-ray diffraction studies of recombinant barley lectin and pro-barley lectin.

Barley lectin (BL) and its precursor form (proBL), synthesized and over-expressed in Escherichia coli, have been crystallized under conditions identical to those used for the closely related lectin wheat germ agglutinin. These lectins are members of the Gramineae family and possess a unique disulfide-rich domain structure. The pro-lectin polypeptides are extended by 15 amino acid residues at the carboxy terminus. This pro-peptide, which is proteolytically removed as the mature lectin is deposited in the vacuoles, is thought to function as a targeting signal for molecular sorting. Crystals of BL and proBL are well ordered and belong to space groups C222(1) and P2(1)2(1)2(1). The unit cell dimensions for BL and proBL are a = 51.9 A, b = 73.7 A, c = 89.3 A (one monomer per asymmetric unit), and a = 45.2 A, b = 70.5 A, c = 111.6 A (two monomers per asymmetric unit), respectively. Diffraction patterns on precession photographs of BL crystals are closely similar to those of mature wheat germ agglutinin crystals, suggesting similar crystal packing and correct conformation of this recombinant protein in terms of the four structural domains and 16 disulfide bridges.

Prohevein is poorly processed but shows enhanced resistance to a chitin-binding fungus in transgenic tomato plants.

In latex of rubber tree (Hevea brasiliensis), prohevein, homologous to potato win gene-encoded proteins, is processed to yield mature hevein. This mature hevein is composed of one chitin-binding domain and the C-terminal polypeptide homologous to pathogenesis-related proteins such as tobacco PR-4 and tomato P2 proteins. In contrast, prohevein was poorly cleaved to form the C-terminal polypeptide in transgenic tomato plants expressing hevein gene (HEV1)-driven polypeptides. However, mature hevein, the N-terminal cleavage form, was not found in this system. Immunoblot analysis of extracellular and intracellular fluid proteins showed that HEV1-encoded polypeptides accumulated intracellularly. In addition, retardation of growth of Trichoderma hamatum was observed in transgenic tomatoes constitutively expressing HEV1-encoded proteins.

Protein targeting to the plant vacuole--a historical perspective.

Although many properties of the targeting of plant endomembrane proteins are similar to mammalian and yeast systems, several clear differences are found that will be stressed in this review. In the past few years, we have seen an advancement in our understanding of the signals for vacuolar protein targeting and some insights into the mechanisms of transport to the vacuole in the plant cell. This work will form the basis for elucidation of the fundamental principles that govern protein trafficking through the secretory system to the vacuole.

Specific binding of nuclear localization sequences to plant nuclei.

We have begun to dissect the import apparatus of higher plants by examining the specific association of nuclear localization sequences (NLSs) with purified plant nuclei. Peptides to the simian virus 40 (SV40) large T antigen NLS and a bipartite NLS of maize were allowed to associate with tobacco and maize nuclei. Wild-type NLSs were found to compete for a single class of low-affinity binding sites having a dissociation constant (Kd) of approximately 200 microM. Peptides to mutant NLSs, which are inefficient in stimulating import, were poor competitors, as were reverse wild-type and non-NLS peptides. The NLS binding site was proteinaceous and resistant to extraction under conditions where pores were still associated. In addition, immunofluorescence and immunoelectron microscopy indicated that binding was at the nuclear envelope. Overall, plant nuclei may be an excellent system to identify components of the import apparatus.

The barley lectin carboxyl-terminal propeptide is a vacuolar protein sorting determinant in plants.

We have previously shown that the 15-amino acid carboxyl-terminal propeptide of probarley lectin is necessary for the proper sorting of this protein to the plant vacuole. A mutant form of the protein lacking the carboxyl-terminal propeptide is secreted. To test whether the carboxyl-terminal propeptide is the vacuole sorting determinant of probarley lectin, we examined in transgenic tobacco the processing and sorting of a series of fusion proteins containing the secreted protein, cucumber chitinase, and regions of probarley lectin. Pulse-labeling experiments demonstrated that the fusion proteins were properly translocated through the tobacco secretory system and that cucumber chitinase and cucumber chitinase fusion proteins lacking the carboxyl-terminal propeptide were secreted. The cucumber chitinase fusion protein containing the carboxyl-terminal propeptide was properly processed and sorted to the vacuole in transgenic tobacco as confirmed by organelle fractionation and electron microscopy immunocytochemistry. Therefore, the barley lectin carboxyl-terminal propeptide is both necessary and sufficient for protein sorting to the plant vacuole.

Protein import into the nucleus: an integrated view.

The directed movement of macromolecules into and out of the nucleus is a fundamental process in eukaryotes and occurs through the nuclear pore complex (NPC). A diverse array of molecules are transported across the nuclear envelope including proteins, mRNAs, tRNAs, snRNP complexes, ribosomal subunits, and in specialized cases, DNA. The structural and functional differences between these molecules point to the mechanistic complexity of NPCs and other components of the nuclear transport apparatus. This machinery must not only recognize within transported molecules specific targeting signals that differ between proteins, RNA, and RNA/protein complexes, it must translocate these molecules across the nuclear envelope. Additional levels of complexity are necessary because molecules such as proteins may continually undergo bidirectional transport across the envelope. Beyond these basic functions, the nuclear transport apparatus is regulated at the level of individual substrates and at more global levels such as coupling to cell cycle regression.

A carboxy-terminal plant vacuolar targeting signal is not recognized by yeast.

Three different classes of signals for plant vacuolar targeting have been defined. Previous work has demonstrated that the carboxyl-terminal propeptide (CTPP) of barley lectin (BL) is a vacuolar targeting signal in tobacco plants. When a mutant BL protein lacking the CTPP is expressed in tobacco, the protein is secreted. In an effort to determine the universality of this signal, the CTPP was tested for its ability to target proteins to the vacuole of Saccharomyces cerevisiae. Genes encoding fusion proteins between the yeast secreted protein invertase and BL domains were synthesized and transformed into an invertase deletion mutant of yeast. Invertase assays on intact and detergent-solubilized cells demonstrated that invertase+CTPP was secreted, while nearly 90% of the invertase::BL+CTPP (fusion protein between invertase and BL containing the CTPP) and invertase::BL-CTPP proteins (fusion between invertase and BL lacking the CTPP) were retained intracellularly. These fusions were secreted in a mutant of yeast that normally secretes proteins targeted to the vacuole. With this and previous work, proteins representing all three classes of plant vacuolar targeting signals have now been tested in yeast, and in all cases, the experiments indicate that the plant proteins are directed to the yeast vacuole using signals other than those recognized by plants.

The basic domain in the bZIP regulatory protein Opaque2 serves two independent functions: DNA binding and nuclear localization.

For organisms to grow and develop, transcriptional regulatory proteins must localize to the nucleus. This movement is mediated by nuclear localization sequences (NLSs) which consist of short stretches of basic amino acids that are part of the structure of mature nuclear proteins. Two NLSs have been previously identified in the maize regulatory protein Opaque2 (O2), a member of the basic-domain, leucine-zipper (bZIP) class of proteins. In this report, it has been determined that both of these NLSs are necessary for import of O2, and the focus has been on a functional analysis of the more efficient, bipartite NLS that is present in the basic or DNA-binding domain. A mutation which contains altered amino acids on both parts of this NLS severely reduced nuclear targeting, and led to the definition of two classes of bipartite NLSs. Since the bipartite NLS is located in the highly conserved. DNA-binding domain, the dual role of this domain was examined using the maize mutant o2-676, in which a conservative mutation eliminates the DNA-binding function. The o2-676 protein localized to the nucleus in maize and the bZIP domain from the mutant protein was sufficient to redirect a reporter protein to the nucleus in transgenic plant cells. Thus, it was possible to show that the nuclear targeting function of this domain is independent of DNA binding. Sequence conservation of the basic domain of other bZIP proteins suggests that the bifunctionality of this domain may be conserved in all members of this class; a consensus sequence for a bipartite NLS in bZIP proteins is suggested.