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.     

Interaction of a potential vacuolar targeting receptor with amino- and carboxyl-terminal targeting determinants.

A protein of 80 kD from developing pea (Pisum sativum) cotyledons has previously been shown to exhibit characteristics of a vacuolar targeting receptor by means of its affinity for the amino-terminal vacuolar targeting sequence of proaleurain from barley (Hordeum vulgare). In this report we show that the same protein also binds to the amino-terminal targeting peptide of prosporamin from sweet potato (Ipomoea batatas) and to the carboxyl-terminal targeting determinant of pro-2S albumin from Brazil nut (Bertholletia excelsa). The receptor protein does not bind to the carboxyl-terminal propeptide (representing the targeting sequence) of barley lectin. The binding of the 80-kD protein to the sporamin determinant involves a motif (NPIR) that has been shown to be crucial for vacuolar targeting in vivo. The binding to the carboxyl-terminal targeting determinant of pro-2S albumin appears to involve the carboxyl-terminal propeptide and the adjacent five amino acids of the mature protein. The 80-kD protein does not bind to peptide sequences that have been shown to be incompetent in directing vacuolar targeting.     

Isolation and characterization of pro-barley lectin expressed in Escherichia coli.

Lectins are a class of proteins with specific carbohydrate-binding properties found in a wide variety of plants and animals. Gramineae lectins are presumably defense-related proteins in plants that exert their effect by binding to N-acetylglucosamine. Barley lectin is a vacuolar protein synthesized with an amino-terminal signal sequence for entering the secretory pathway and a carboxyl-terminal propeptide necessary for proper targeting to the vacuole. To analyze the three-dimensional structure of barley lectin with the carboxyl-terminal extension and to investigate whether the conversion of the prolectin into the mature molecule leads to a conformational change, the precursor and the mature forms of barley lectin were expressed in Escherichia coli. Both proteins accumulated in denatured form in inclusion bodies were solubilized in 8 M urea and renatured in a redox buffer system. Active pro- and mature barley lectins were purified to homogeneity by affinity chromatography.     

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.     

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.     

Determination of the functional elements within the vacuolar targeting signal of barley lectin.

We have previously demonstrated that the carboxyl-terminal propeptide of barley lectin is both necessary and sufficient for protein sorting to the plant vacuole. Specific mutations were constructed to determine which amino acid residues or secondary structural determinants of the carboxyl-terminal propeptide affect proper protein sorting. We have found that no consensus sequence or common structural determinants are required for proper sorting of barley lectin to the vacuole. However, our analysis demonstrated the importance of hydrophobic residues in vacuolar targeting. In addition, at least three exposed amino acid residues are necessary for efficient sorting. Sorting was disrupted by the addition of two glycine residues at the carboxyl-terminal end of the targeting signal or by the translocation of the glycan to the carboxy terminus of the propeptide. These results suggest that some components of the sorting apparatus interact with the carboxy terminus of the propeptide.     

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and -1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.     

Post-translational maturation of natural and drug-induced missorted phytohemagglutinin

The bean lectin phytohemagglutinin (PHA) was expressed in transgenic suspension-cultured BY-2 tobacco cells simultaneously with another recombinant vacuolar protein, the sweet potato sporamin. In contrast to previous observations in different transgenic plant systems when expressed in BY-2 tobacco cells, phytohemagglutinin is mostly but not exclusively targeted to the vacuole. Indeed, a small amount of recombinant phytohemagglutinin is secreted into the culture medium of tobacco cells. Furthermore part of this extracellular phytohemagglutinin has no lectin activity and presents an abnormal glycosylation consistent with higher accessibility of glycans N-linked to these extracellular phytohemagglutinin forms. Phytohemagglutinin secretion occurs regardless of recombinant protein expression level. Consequently, missorting in this case is due to an abnormal phytohemagglutinin conformation or oligomerization rather than to receptor saturation. The treatment of BY-2 cells with drugs, such as monensin and wortmannin, increases even more the transport of phytohemagglutinin to the cell surface through a general inhibition of the sorting mechanisms of vacuolar proteins. The sensitivity to wortmannin is similar for the sorting of phytohemagglutinin and endogenous tobacco chitinase and β-1,3-glucanase, suggesting that phytohemagglutinin and COOH-terminal propeptide mediated vacuolar sorting share similar mechanisms. A characterization of glycans N-linked to extracellular phytohemagglutinin secreted by monensin- or wortmannin-treated transgenic tobacco cells illustrates that in contrast with monensin, wortmannin completely inhibits the sorting of vacuolar proteins without having any effect on the efficiency of Golgi processing enzymes.     

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.     

Antimicrobial peptides fromMirabilis jalapa andAmaranthus caudatus: expression, processing, localization and biological activity in transgenic tobacco

The cDNAs encoding the seed antimicrobial peptides (AMPs) fromMirabilis jalapa (Mj-AMP2) andAmaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolleet al., Plant Mol Biol 28:713–721 (1995) and 22:1187–1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195–1206 (1991)]; an Ac-AMP2wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing eitherwild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. Thein vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with eitherBotrytis einerea orAlternaria longipes.     

A carboxyl-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco.

Barley lectin is synthesized as a preproprotein with a glycosylated carboxyl-terminal propeptide (CTPP) that is removed before or concomitant with deposition of the mature protein in vacuoles. Expression of a cDNA clone encoding barley lectin in transformed tobacco plants results in the correct processing, maturation, and accumulation of active barley lectin in vacuoles [Wilkins, T.A., Bednarek, S.Y., and Raikhel, N.V. (1990). Plant Cell 2, 301-313]. The glycan of the propeptide is not essential for vacuolar sorting, but may influence the rate of post-translational processing [Wilkins, T.A., Bednarek, S.Y., and Raikhel, N.V. (1990). Plant Cell 2, 301-313]. To investigate the functional role of the CTPP in processing, assembly, and sorting of barley lectin to vacuoles, a mutant barley lectin cDNA clone lacking the 15-amino acid CTPP was prepared. The CTPP deletion mutant of barley lectin was expressed in tobacco protoplasts, suspension-cultured cells, and transgenic plants. In all three systems, the wild-type barley lectin was sorted to vacuoles, whereas the mutant barley lectin was secreted to the incubation media. Therefore, we conclude that the carboxyl-terminal domain of the barley lectin proprotein is necessary for the efficient sorting of this protein to plant cell vacuoles.     

The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the ER

Proteins are co-translationally transferred into the endo-plasmic reticulum (ER) and then either retained or transported to different intracellular compartments or to the extracellular space. Various molecular signals necessary for retention in the ER or targeting to different compartments have been identified. In particular, the HDEL and KDEL signals used for retention of proteins in yeast and animal ER have also been described at the C-terminal end of soluble ER processing enzymes in plants. The fusion of a KDEL extension to vacuolar proteins is sufficient for their retention in the ER of transgenic plant cells. However, recent results obtained using the same strategy indicate that HDEL does not contain sufficient information for full retention of phaseolin expressed in tobacco. In the present study, an HDEL C-terminal extension was fused to the vacuolar or extracellular (Δpro) forms of sporamin. The resulting SpoHDEL or ΔproHDEL, as well as Spo and Δpro, were expressed at high levels in transgenic tobacco cells ( Nicotiana tabacum cv BY2). The intracellular location of these different forms of recombinant sporamin was studied by subcellular fractionation. The results clearly indicate that addition of an HDEL extension to either Spo or Δpro induces accumulation of these sporamin forms in a compartment that co-purifies with the ER markers NADH cytochrome C reductase, binding protein (BiP) and calnexin. In addition, a significant SpoHDEL or ΔproHDEL fraction that escapes the ER retention machinery is transported to the vacuole. From these results, it may be proposed that, in addition to its function as an ER retention signal, HDEL could also act in quality control by targeting chaperones or chaperone-bound proteins that escape the ER to the plant lysosomal compartment for degradation.     

Transport of a Sweet Potato Storage Protein, Sporamin, to the Vacuole in Yeast Cells

The propeptide of a precursor to sporamin, a storage proteinof sweet potato, is required for targeting of sporamin to thevacuole in transformed tobacco cells (Matsuoka and Nakamura1991). A fusion gene consisting of an inducible GAL 10 promoterand sporamin cDNA was introduced into Saccharomyces cerevisiaeby use either of a multiple-copy plasmid (YEpSAD16) or of asingle-copy plasmid (YCpSAD16) to control the level of expressionof the precursor. Although we could not detect any sporamin-relatedpolypeptides in cells that harbored YCpSAD16, extracts fromcells that harbored YEpSAD16 contained multiple forms of sporaminrelatedpolypeptides: preprosporamin, prosporamin and several polypeptidesthat were smaller than prosporamin. However, YCpSAD16 directedthe accumulation of prosporamin in pep4 mutant yeast cells thatlack vacuolar proteases, andpep4 mutant cells that harboredYEpSAD16 did not contain any sporamin-related polypeptides smallerthan prosporamin. The vacuole fractions isolated from the wild-typeand pep4 mutant cells contained sporamin-related polypeptidessmaller than prosporamin and prosporamin, respectively. Theseand other results suggest that, at a low level of expressionof the precursor, prosporamin is transported to the vacuoleand degraded by vacuolar proteases. A mutant precursor to sporamin,in which the propeptide and the N-terminal region of maturesporamin were replaced by an unrelated sequence of four aminoacid residues, directed the secretion of sporamin to the culturemedium in transformed tobacco cells. However, this mutationdid not affect the transport of sporamin to the vacuole in yeastcells and none of the sporamin-related polypeptides were secretedto the extracellular space. (Received July 16, 1991; Accepted March 25, 1992)     

Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway

In Saccharomyces cerevisiae the vacuolar protein aminopeptidase I (API) is localized to the vacuole independent of the secretory pathway. The alternate targeting mechanism used by this protein has not been characterized. API is synthesized as a 61-kD soluble cytosolic precursor. Upon delivery to the vacuole, the amino-terminal propeptide is removed by proteinase B (PrB) to yield the mature 50-kD hydrolase. We exploited this delivery-dependent maturation event in a mutant screen to identify genes whose products are involved in API targeting. Using antiserum to the API propeptide, we isolated mutants that accumulate precursor API. These mutants, designated cvt, fall into eight complementation groups, five of which define novel genes. These five complementation groups exhibit a specific defect in maturation of API, but do not have a significant effect on vacuolar protein targeting through the secretory pathway. Localization studies show that precursor API accumulates outside of the vacuole in all five groups, indicating that they are blocked in API targeting and/or translocation. Future analysis of these gene products will provide information about the subcellular components involved in this alternate mechanism of vacuolar protein localization.     

Proaleurain vacuolar targeting is mediated by short contiguous peptide interactions.

Targeting of soluble proteins to the plant vacuole is mediated by determinants that reside in the polypeptide. We identified the vacuolar targeting determinant of aleurain, a plant vacuolar thiol protease, by incorporating different sequences from proaleurain into the secreted thiol protease, proendoproteinase B (proEP-B), and vice versa. The targeting fates of the chimeric proteins were analyzed by transient expression in electroporated tobacco protoplasts. The targeting determinant SSSSFADSNPIR is positioned at the N terminus of the aleurain propeptide, and its substitution into the propeptide of EP-B caused vacuolar targeting of the resulting chimeric protein. This determinant can be divided into two smaller determinants, SSSSFADS and SNPIR, each of which is sufficient to target proEP-B chimeras to the vacuole, but with lower efficiency. These smaller determinants interact in a positive manner because the combined determinant SSSSFADSNPIR targeted proEP-B with an efficiency greater than each of the smaller determinants alone. Accordingly, the efficiency of aleurain targeting was decreased when either of the smaller determinants was disrupted by replacement with similarly positioned proEP-B sequences. Further experiments on proaleurain identified an additional determinant, VTDRAAST, adjacent to the SSSSFADSNPIR determinant that is also necessary for efficient vacuolar targeting. Our results provide evidence that efficient vacuolar targeting of this thiol protease in plant cells is mediated by the combined action of smaller contiguous determinants; two of these alone are sufficient for vacuolar targeting.     

Analysis of sporamin forms expressed in different subcellular compartments of transgenic tobacco plants by IMAC and ESI-MS

Various forms of sporamin (spo, Δpro, ΔproHDEL and spoHDEL) corresponding to different targetings within the plant cell, respectively, to the vacuole, the extracellular compartment and the endoplasmic reticulum (ER) for the last two were used as model proteins to study further structural modifications. These proteins were expressed in tobacco plants (Nicotiana tabacum L. cv. PB D6), purified by immobilized metal ion affinity chromatography (IMAC) and then analyzed by electrospray ionization mass spectrometry (ESI-MS). Conformational and post-translational modifications were observed between these different forms of sporamin, extracted from leaves. By the combination of these two techniques, detection of discrete intermediate species was achieved. Three distinct entities were detected for the vacuolar form of sporamin (spo) indicating a two-step processing of the protein, while only one entity, shorter by two amino-acids at its N-terminus, was detected for the extracellular form of sporamin (Δpro). The analysis of sporamin forms with an HDEL amino-acid extension can not be deduced easily from MS data and may reflect post-translational modifications distinct from proteolytic processing. Thus post-translational modifications appear to be closely related to targeting within the plant cell.     

Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway

The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole. API lacks a standard signal sequence and contains an unusual amino-terminal propeptide. We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole. API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner. The half-time for processing is approximately 45 min. The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway. The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner. Neither the precursor nor mature form of API are secreted into the extracellular fraction in vps mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway. Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein. These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.     

Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles

Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion.     

Yeast carboxypeptidase Y vacuolar targeting signal is defined by four propeptide amino acids

The amino-terminal propeptide of carboxypeptidase Y (CPY) is necessary and sufficient for targeting this glycoprotein to the vacuole of Saccharomyces cerevisiae. A 16 amino acid stretch of the propeptide was subjected to region-directed mutagenesis using randomized oligonucleotides. Mutations altering any of four contiguous amino acids, Gln-Arg-Pro-Leu, resulted in secretion of the encoded CPY precursor (proCPY), demonstrating that these residues form the core of the vacuolar targeting signal. Cells that simultaneously synthesize both wild-type and sorting-defective forms of proCPY efficiently sort and deliver only the wild-type molecule to the vacuole. These results indicate that the PRC1 missorting mutations are cis-dominant, implying that the mutant forms of proCPY are secreted as a consequence of failing to interact with the sorting apparatus, rather than a general poisoning of the vacuolar protein targeting system.