Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information.

An inactive precursor form of proteinase A (PrA) transits through the early secretory pathway before final vacuolar delivery. We used gene fusions between the gene coding for PrA (PEP4) and the gene coding for the secretory enzyme invertase (SUC2) to identify vacuolar protein-sorting information in the PrA precursor. We found that the 76-amino-acid preprosegment of PrA contains at least two sorting signals: an amino-terminal signal peptide that is cleaved from the protein at the level of the endoplasmic reticulum followed by the prosegment which functions as a vacuolar protein-sorting signal. PrA-invertase hybrid proteins that carried this sequence information were accurately sorted to the yeast vacuole as determined by cell fractionation and immunolocalization studies. Hybrid proteins lacking all or a portion of the PrA prosegment were secreted from the cell. Our gene fusion data together with an analysis of the wild-type PrA protein indicated that N-linked carbohydrate modifications are not required for vacuolar sorting of this protein. Furthermore, results obtained with a set of deletion mutations constructed in the PrA prosegment indicated that this sequence also contributes to proper folding of this polypeptide into a stable transit-competent molecule.     

Crystal structure of aspartic proteinase from Irpex lacteus in complex with inhibitor pepstatin

The crystal structure of Irpex lacteus aspartic proteinase (ILAP) in complex with pepstatin (a six amino acid residue peptide-like inhibitor) was determined at 1.3A resolution. ILAP is a pepsin-like enzyme, widely distributed in nature, with high milk-clotting activity relative to proteolytic activity. The overall structure was in good topological agreement with pepsin and other aspartic proteases. The structure and interaction pattern around the catalytic site were conserved, in agreement with the other aspartic proteinase/inhibitor complex structures reported previously. The high-resolution data also supported the transition state model, as proposed previously for the catalytic mechanism of aspartic proteinase. Unlike the other aspartic proteinases, ILAP was found to require hydrophobic residues either in the P(1) or P(1') site, and also in the P(4) and/or P(3) site(s) for secondary interactions. The inhibitor complex structure also revealed the substrate binding mechanism of ILAP at the P(3) and P(4) site of the substrate, where the inserted loop built up the unique hydrophobic pocket at the P(4) site.     

The aspartic proteinase of barley is a vacuolar enzyme that processes probarley lectin in vitro.

Previous work suggested that the aspartic proteinase from Hordeum vulgare (HvAP) would be a vacuolar protein in plant cells. Based on N-terminal sequencing we show that the in vitro-translated protein was translocated into the lumen of microsomal membranes, causing a concomitant removal of 25 amino acid residues from the protein. Vacuoles were purified from barley leaf protoplasts and were shown to contain all of the aspartic proteinase activity found in the protoplasts. This vacuolar localization of HvAP was confirmed with immunocytochemical electron microscopy using antibodies to HvAP in both barley leaf and root cells. In an attempt to discern a function for this protease, we investigated the ability of HvAP to process the C-terminal proregion of barley lectin (BL) in vitro. Prolectin (proBL), expressed in bacteria, was processed rapidly when HvAP was added. Using several means, we were able to determine that 13 amino acid residues at the C terminus of proBL were cleaved off, whereas the N terminus stayed intact during this incubation. Immunohistochemical electron microscopy showed that HvAP and BL are co-localized in the root cells of developing embryos and germinating seedlings. Thus, we propose that the vacuolar HvAP participates in processing the C terminus of BL.     

Crystal structure of cardosin A, a glycosylated and Arg-Gly-Asp-containing aspartic proteinase from the flowers of Cynara cardunculus L.

Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Manbeta4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two beta-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed.     

Membrane anchors for vacuolar targeting: application in plant bioreactors

Transgenic plants are attractive expression systems for producing recombinant proteins. Plant cells compartmentalize and store metabolites and proteins in vacuoles, but foreign proteins need to be targeted to the correct compartments if they are to accumulate in a stable fashion. Here we present a general strategy in which unique transmembrane and cytoplasmic tail sequences are used as anchors for delivering recombinant proteins via distinct vesicular transport pathways to specific vacuolar compartments where stable accumulation can occur.     

Cloning and characterization of cDNA encoding cardosin A, an RGD-containing plant aspartic proteinase.

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth.     

Processing and trafficking of a single isoform of the aspartic proteinase cardosin A on the vacuolar pathway

Cardosin A is the major vacuolar aspartic proteinase (APs) (E.C.3.4.23) in pistils of Cynara cardunculus L. (cardoon). Plant APs carry a unique domain, the plant-specific-insert (PSI), and a pro-segment which are separated from the catalytic domains during maturation but the sequence and location of processing steps for cardosins have not been established. Here transient expression in tobacco and inducible expression in Arabidopsis indicate that processing of cardosin A is conserved in heterologous species. Pulse chase analysis in tobacco protoplasts indicated that cleavage at the carboxy-terminus of the PSI could generate a short-lived 50 kDa intermediate which was converted to a more stable 35 kDa intermediate by removal of the PSI. Processing intermediates detected immunologically in tobacco leaves and Arabidopsis seedlings confirmed that cleavage at the amino-terminus of the PSI either preceded or followed quickly after cleavage at its carboxy-terminus. Thus removal of PSI preceded the loss of the prosegment in contrast to the well-characterised barley AP, phytepsin. PreprocardosinA acquired a complex glycan and its processing was inhibited by brefeldin A and dominant-inhibitory AtSAR1 or AtRAB-D2^a mutants indicating that it was transported via the Golgi and that processing followed ER export. The 35 kDa intermediate was present in the cell wall and protoplast culture medium as well as the vacuole but the 31 kDa mature subunit, lacking the amino-terminal prosegment, was detected only in the vacuole. Thus maturation appears to occur only after sorting from the trans-Golgi to the vacuole. Processing or transport of cardosin A was apparently slower in tobacco protoplasts than in whole cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Phytepsin, a barley vacuolar aspartic proteinase, is highly expressed during autolysis of developing tracheary elements and sieve cells

Vacuolarisation, formation of autophagocytotic vacuoles and tonoplast disruption have been reported in plant cells undergoing developmentally regulated programmed cell death (PCD), but little is known about the vacuolar proteins involved. In HeLa cells, cathepsin D, a lysosomal aspartic proteinase has been shown to mediate PCD. Based on immunohistochemical staining of barley roots, we show here that the previously well characterised barley vacuolar aspartic proteinase (phytepsin), a plant homologue to cathepsin D, is highly expressed both during formation of tracheary elements and during partial autolysis of sieve cells. In serial transverse sections of the vascular cylinder, starting from the root tip, phytepsin is expressed in root cap cells, in the tracheary elements of early and late metaxylem, and in the sieve cells of the protophloem and metaphloem. Aleurain, a barley vacuolar cysteine proteinase, is expressed similarly in root cap cells but differently in the tracheary elements of protoxylem and early metaxylem. This is the first evidence that a vacuolar aspartic proteinase, in analogy to cathepsin D in animals, may play a role in the active autolysis of plant cells.     

Cold inactivation of vacuolar proton-ATPases

Incubation of the reconstituted H+-ATPase from chromaffin granules on ice resulted in inactivation of the proton-pumping and ATPase activities of the enzyme. Inactivation was dependent on the presence of Mg2+, Cl-, and ATP during the incubation at low temperature. Approximately 1 mM ATP, 1 mM Mg2+, and 200 mM Cl- were required for maximum inactivation. Incubation for about 10 min on ice was required to achieve 50% inactivation. A much smaller decline in activity was observed when the enzyme was incubated at room temperature with the same chemicals. Inactivation in the cold resulted in the release of five polypeptides from the membrane with apparent molecular masses of 72, 57, 41, 34, and 33 kDa on sodium dodecyl sulfate gels. Three of the polypeptides of 72, 57, and 34 kDa were identified as subunits of vacuolar H+-ATPases by antibody cross-reactivity. Similar results were obtained with several other vacuolar H+-ATPases including those from plant sources. It was concluded that the catalytic sector of the enzyme is released from the H+-ATPase complex by cold treatment, resulting in inactivation of the enzyme.     

Crystal structure of plant asparaginase

In plants, specialized enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. In addition, K+-independent plant asparaginases are also active in splitting the aberrant isoaspartyl peptide bonds, which makes these proteins important for seed viability and germination. Here, we present the crystal structure of potassium-independent L-asparaginase from yellow lupine (LlA) and confirm the classification of this group of enzymes in the family of Ntn-hydrolases. The alpha- and beta-subunits that form the mature (alphabeta)2 enzyme arise from autoproteolytic cleavage of two copies of a precursor protein. In common with other Ntn-hydrolases, the (alphabeta) heterodimer has a sandwich-like fold with two beta-sheets flanked by two layers of alpha-helices (alphabetabetaalpha). The nucleophilic Thr193 residue, which is liberated in the autocatalytic event at the N terminus of subunit beta, is part of an active site that is similar to that observed in a homologous bacterial enzyme. An unusual sodium-binding loop of the bacterial protein, necessary for proper positioning of all components of the active site, shows strictly conserved conformation and metal coordination in the plant enzyme. A chloride anion complexed in the LlA structure marks the position of the alpha-carboxylate group of the L-aspartyl substrate/product moiety. Detailed analysis of the active site suggests why the plant enzyme hydrolyzes asparagine and its beta-peptides but is inactive towards substrates accepted by similar Ntn-hydrolases, such as taspase1, an enzyme implicated in some human leukemias. Structural comparisons of LlA and taspase1 provide interesting insights into the role of small inorganic ions in the latter enzyme.     

Expression of biotin-binding proteins,avidin and streptavidin,in plant tissues using plant vacuolar targeting sequences

Tobacco plants have been developed which constitutively express high levels of the biotin-binding proteins, avidin and streptavidin. These plants were phenotypically normal and produced fertile pollen and seeds. The transgene was expressed and its product located in the vacuoles of most cell types in the plants. Targeting was achieved by use of N-terminal vacuolar targeting sequences derived from potato proteinase inhibitors which are known to target constitutively to vacuoles in potato tubers and, under wound-induction, in tomato leaves. Avidin was located in protein body-like structures within the vacuole and transgene protein levels remained relatively constant throughout the lifetime of the leaf. We describe two chimeric constructs with similar levels of expression. One comprised a potato proteinase inhibitor I signal peptide cDNA sequence attached to an avidin cDNA and the second a potato proteinase inhibitor II signal peptide genomic sequence (including an intron) attached to a core streptavidin synthetic sequence. We were unable to regenerate plants when transformation used constructs lacking the targeting sequences. The highest levels observed (up to 1.5% of total leaf protein) confirm the vacuole as the organelle of choice for stable storage of plant-toxic transgene products. The efficient targeting of these proteins did not result in any measured changes in plant biotinmetabolism.     

Crystal structure of colicin E3: implications for cell entry and ribosome inactivation.

Colicins kill E. coli by a process that involves binding to a surface receptor, entering the cell, and, finally, intoxicating it. The lethal action of colicin E3 is a specific cleavage in the ribosomal decoding A site. The crystal structure of colicin E3, reported here in a binary complex with its immunity protein (IP), reveals a Y-shaped molecule with the receptor binding domain forming a 100 A long stalk and the two globular heads of the translocation domain (T) and the catalytic domain (C) comprising the two arms. Active site residues are D510, H513, E517, and R545. IP is buried between T and C. Rather than blocking the active site, IP prevents access of the active site to the ribosome.     

Structure and function of plant aspartic proteinases.

Aspartic proteinases of the A1 family are widely distributed among plant species and have been purified from a variety of tissues. They are most active at acidic pH, are specifically inhibited by pepstatin A and contain two aspartic residues indispensible for catalytic activity. The three-dimensional structure of two plant aspartic proteinases has been determined, sharing significant structural similarity with other known structures of mammalian aspartic proteinases. With a few exceptions, the majority of plant aspartic proteinases identified so far are synthesized with a prepro-domain and subsequently converted to mature two-chain enzymes. A characteristic feature of the majority of plant aspartic proteinase precursors is the presence of an extra protein domain of about 100 amino acids known as the plant-specific insert, which is highly similar both in sequence and structure to saposin-like proteins. This insert is usually removed during processing and is absent from the mature form of the enzyme. Its functions are still unclear but a role in the vacuolar targeting of the precursors has been proposed. The biological role of plant aspartic proteinases is also not completely established. Nevertheless, their involvement in protein processing or degradation under different conditions and in different stages of plant development suggests some functional specialization. Based on the recent findings on the diversity of A1 family members in Arabidopsis thaliana, new questions concerning novel structure-function relationships among plant aspartic proteinases are now starting to be addressed.     

Evidence for the presence of proteinase inhibitor I in vacuolar protein bodies of plant cells

Summary Protein bodies induced in tomato leaf cells by wounding were shown to contain proteinase Inhibitor I by using ferritin-labelled antibodies, fluorescein-labelled antibodies, and cytochrome C-labelled antibody fragments. Both pre-embedding and postembedding techniques were used. Nonspecific binding was least when p-formaldehyde was used as the initial fixative followed by treatment with cytochrome c-labelled antibody fragments.Abbreviations Fab antibody fragments - BSA bovine serum albumin - GMA glycol methacrylate - THB Tris-HCl buffer Taken in part from a doctoral (Ph.D.) dissertation submitted to Washington State University by Vivian V. Yang. This work was supported largely by NSF Grant GB-29614X (LKS) and in part by the United States Department of Agricultural Cooperative States Research Service Grant 316-15-30 (CAR), the National Science Foundation Grant GB-37972 (CAR), and the College of Agriculture Research Center, Washington State University, Pullman, WA 99163, Scientific Paper No. 4525, Project 1791.Program in Genetics and Department of Botany. To whom reprint requests should be sent.Department of Agricultural Chemistry and Program in Biochemistry and Biophysics.     

Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens.

The Saccharomyces cerevisiae PEP4 gene encodes proteinase A, an aspartyl protease. pep4 mutants are defective in the activation of many vacuolar hydrolases, including proteinase B. We have expressed a pep4 mutation which directs the accumulation of pro-proteinase A with a defective active site. Co-expression with PEP4 leads to normal processing, i.e. the mutant zymogen is functional as a substrate for the maturation reaction in trans. We conclude that wild-type pro-proteinase A has the ability to mediate its own activation. Elimination of the co-expressed PEP4 gene did not effectively stop the processing of the mutant zymogen, owing to a strong, proteinase-B-dependent, phenotypic lag. In a proteinase-B-negative strain, processing of pro-proteinase A led to an active form of a higher molecular mass than the normal mature form.     

The structure of plant vacuolar membranes according to infrared spectroscopy

The structure of the vacuolar membrane (tonoplast) was studied in red beet roots by IR spectroscopy. The vacuolar membrane was shown to be composed of highly ordered lipids which form regions of free liquid lipid bilayer loosely bound to integral proteins. The prevalence of polar lipids in the tonoplast is responsible for the high elasticity and fluidity of the membrane. The presence of alpha-tocopherol in the tonoplast membrane accounts for a high antioxidant activity of the membrane. Integral proteins are immersed into the liquid matrix of the lipid bilayer to a different extent. Examination of the temperature effect on the kinetics of the hydrogen-deuterium exchange in integral membrane proteins showed that the efficient energy of the hydrogen exchange activation was 24 +/- 4 kcal/mol at 19-40 degrees C and increased to 54 kcal/mol at 40-50 degrees C because of the thermal denaturation of proteins. The secondary structure of integral membrane proteins is characterized by a high content of alpha-helices (53%) which decreased to 8% after the extraction of lipids.     

Plant-specific insertions in the soybean aspartic proteinases, soyAP1 and soyAP2, perform different functions of vacuolar targeting

Most aspartic proteinases (APs) of plant origin are characterized by the presence of plant-specific insertion (PSI) in their primary structure. PSI has been reported to function as signals for both transport of AP molecules from the endoplasmic reticulum (ER) and for their targeting to the vacuole. To determine the functions of the PSIs in soyAP1 and soyAP2 identified in our previous study, we examined their subcellular localization by transient expression of a green fluorescent protein (GFP) fusion protein in the protoplasts of Arabidopsis suspension-cultured cells. Both soyAP1-GFP and soyAP2-GFP were targeted to the vacuole. To confirm the role of the PSI, we prepared PSI-deleted soyAP1 and soyAP2, and investigated their vacuolar targeting by the same method. While the former deletion mutant was always transported to the vacuole, the latter sometimes remained in the ER and was only sometimes transported to the vacuole. These observations indicated that, in the case of soyAP1, the PSI is not involved in vacuolar targeting, also suggesting that the function of the PSI differs depending on its origin.