MCB-9507030 Raikhel The long-term goal of this project is to investigate the molecular mechanisms of protein sorting to plant vacuoles. Three types of plant vacuolar sorting signals have been identified, based on their location within proteins. Signals within the propeptide regions at the C-terminus (CTPPs) and N-terminus (NTPPs) have been characterized as well as signals within the body of mature proteins. Among the different types of signals, there does not appear to be a common sequence motif responsible for vacuolar sorting, and a distinct receptor is implicated in the recognition of each signal. We have characterized the CTPP signals in plant secretory proteins and identified specific mutations that cause secretion of vacuolar proteins. There is no homology between different CTPPs, and various mutations and deletions in CTPP are tolerated by the sorting machinery. Although vacuolar sorting signals and most likely receptors that recognize these signals are unique, the components involved in vesicle transport are common among eukaryotic cells. To this end we have isolated a functional homolog of yeast Pepl2p from Arabidopsis thaliana, an integral protein thought to be involved in the specific recognition of vesicles destined to the vacuole. We plan to characterize the A. thaliana Pepl2p and use it as a tool to isolate other components of the machinery, including the receptors that recognize vacuolar sorting signals. To compliment our molecular and biochemical approaches we have isolated several putative A. thaliana mutants with Altered Vacuolar Sorting (AVS). Based on this the following objectives are proposed: 1) Completely sequence the aPEP12 cDNA clone, raise antibodies against aPepl2p, and use them to determine the subcellular location, by organellar fractionation and electron microscopy. 2) Study the function of aPepl2p in plant cells by transforming aPEP12 in the antisense orientation in to A. thaliana plants expressing independently barley l ectin, sporamin, or phytohemagglutinin, and analyze the effect of altered levels of aPepl2p on sorting of either one or all three different soluble vacuolar proteins by pulse-chase labeling followed by immunoprecipitation and organellar fractionation. 3) Identify components that interact with aPepl2p by gradient centrifugation and purify them by co-immunoprecipitation using aPepl 2p antibodies. Additional approaches will include affinity chromatography or chemical cross-linking. Corresponding genes will eventually be cloned. 4) Isolate avs mutants of Arabidopsis. We shall continue to screen for additional mutants that secrete CTPP-containing proteins and set up a similar screen to isolate mutants that secrete NTPP- containing proteins. Priority will be placed on the isolation of transacting mutations. 5) Characterize avs mutants of Arabidopsis. The mutants isolated for secretion of CTPP containing proteins will be tested for secretion or vacuolar sorting of NTPP-containing proteins (and vice versa) by pulse-chase analysis and organellar purification. Mutantsspecific for either the CTPP or the NTPP pathways will be of particular interest because these have the highest potential to identify components unique to plant vacuolar sorting. However, mutants that affect both pathways could also help us to identify important common components. The research should provide important insights into the fundamental processes of the vacuolar sorting machinery that may be unique to plants or of broad significance. Beyond its contribution to basic knowledge, this work could improve the success rate of sorting novel gene products to desired parts of the cell which might lead to crop improvement. %%% The vacuole is the site of storage of protein and sugars within the plant cell. It is also the site at which many of these storage proteins and sugars are broken down. With regard to this latter function, the vacuole is somewhat similar to the animal cell lysosome. Over the past five years, there has been rapid progress in identifying some of the cellular machinery by which material is trafficked to the iysosome in mammalian cells and yeast. In this study, this machinery is described in plants. The plant machinery is explored starting with a plant protein involved in vacuole traffic which can also work to allow traffic to the yeast vacuole. This protein and the protein and membranes with which it interacts in the plant cell are described. Other proteins and genes important in correctly targeting proteins to plant vacuoles are discovered through generating mutants defective in correct targeting of soluble vacuolar proteins. This enhances our understanding of how traffic to the plant vacuole is regulated. There are many eventual applications of this work, including altering the components of the targeting machinery in order to get more efficient storage and transport of novel proteins to the plant vacuole for the production of enhanced grain and seed crops. ***
