Plant Cells Contain Two Functionally Distinct Vacuolar Compartments

Proteolytic enzymes are known to be associated with developmentally programmed cell death during organ senescence and tracheary element differentiation. Recent evidence also links proteinases with some types of pathogen- and stress-induced cell suicide. The precise roles of proteinases in these and other plant programmed cell death processes are not understood, however. To provide a framework for consideration of the importance of proteinases during plant cell suicide, characteristics of the best-known proteinases from plants including subtilisin-type and papain-type enzymes, phytepsins, metalloproteinases and the 26S proteasome are summarized. Examples of serine, cysteine, aspartic, metallo- and threonine proteinases linked to animal programmed cell death are cited and the potential for plant proteinases to act as mediators of signal transduction and as effectors of programmed cell death is discussed.

Plant proteolytic enzymes: possible roles during programmed cell death

We determined at 2.3 A resolution the crystal structure of prophytepsin, a zymogen of a barley vacuolar aspartic proteinase. In addition to the classical pepsin‐like bilobal main body of phytepsin, we also traced most of the propeptide, as well as an independent plant‐specific domain, never before described in structural terms. The structure revealed that, in addition to the propeptide, 13 N‐terminal residues of the mature phytepsin are essential for inactivation of the enzyme. Comparison of the plant‐specific domain with NK‐lysin indicates that these two saposin‐like structures are closely related, suggesting that all saposins and saposin‐like domains share a common topology. Structural analysis of prophytepsin led to the identification of a putative membrane receptor‐binding site involved in Golgi‐mediated transport to vacuoles.

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Crystal structure of plant aspartic proteinase prophytepsin: inactivation and vacuolar targeting.

Endoproteases of barley and malt

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

Paula Sarkkinen

Papers

Hydrolytic specificity of the barley grain aspartic proteinase.