Vesicle Trafficking From the Trans-Golgi Network to Prevacuolar Compartment in Arabidopsis

Michigan State University, East Lansing MI

Investigators

Abstract

Some mechanisms of protein trafficking through the secretory system are conserved among all eukaryotes; nevertheless, plants possess a highly complex vacuolar targeting machinery. The complexity is the result of the diverse nature of vacuoles in some cell types. At least two distinct types of vacuolar structure have been observed: one with a lytic and one with a storage function. Proteins of the secretory pathway are usually first inserted into the endoplasmic reticulum before being transported through the endomembrane system in small transport vesicles. Vacuolar proteins have signals that sort them to vacuoles; if such signals are deleted, vacuolar proteins are instead secreted from the plant cell. The plant vacuolar sorting signals identified to date fall into three classes: N-terminal propeptides (NTPPs), C-terminal propeptides (CTPPs), and much less characterized portions of mature proteins. A putative vacuolar sorting receptor has been identified in pea (BP80) and in Arabidopsis (AtELP), and it likely functions in the route taken by the proteins with NTPP signals. This putative receptor has been found in the Golgi, trans-Golgi network (TGN)-derived vesicles, and in the prevacuolar compartment (PVC). Although transport between the TGN and PVC is critical for faithful delivery of cargo proteins to the vacuole, very little is known about this process in any multicellular organism. This project will focus on mechanisms that mediate transport between the TGN and PVC. Experiments to be carried out in this project aim at investigating further (1) the mechanisms and diversity of NTPP pathway in plants, (2) the in vivo function of the putative vacuolar sorting receptor, (3) the PVC, and (4) vesicles that deliver cargo to that compartment. Although much work has been done with Arabidopsis, no endogenous vacuolar proteins from this plant have as yet been characterized and no reliable antibodies against vacuolar proteins are available. Dr. Raikhel will prepare and characterize antibodies against vacuolar markers to analyze transport of Arabidopsis proteins to the vacuoles using information available in the Arabidopsis EST database and by purifying proteins from isolated vesicles. These endogenous markers and her laboratory's well-characterized heterologous markers will be used to analyze trafficking in both wild-type and Arabidopsis mutants created in the course of this research. The putative vacuolar sorting receptor, AtELP, is a representative of a small gene family. Characterization of the cargo specificity of AtELP has been done only in in vitro assays; no proteins that bind AtELP in vivo have been identified. One important question is whether AtELP and related proteins interact with a number of different vacuolar sorting signals or only with NTPP-containing cargo. Dr. Raikhel will try to answer this question using experimental strategies that include reverse genetics, biochemistry, and cell biology. The nature and function of the PVC in plants is not well defined. Unlike in yeast, most SNARE proteins that mediate trafficking between the Golgi and PVC in plants are represented by small gene families. In addition, the Principal Investigator has found that in plants, some SNARE proteins have different roles than in yeast. Experiments will address the question of whether this is a result of redundancy or functional complexity of plant SNAREs, and structural and functional diversity of the plant PVCs. They will also examine the cargo and resident proteins of the PVC to see whether only proteins found in the AtELP-carrying vesicles are present in the PVC, or whether the PVC is a compartment where vesicles taking different routes deliver their cargo. They will use immunoprecipitation experiments followed by a proteomic approach to obtain sequence information of cargo proteins from various vesicles and the PVC. This research will provide important insights into the fundamental processes of vacuolar sorting in multicellular organisms that may be unique to plants and are of broad significance. Beyond its contribution to basic knowledge, this work will improve the success rate of sorting novel gene products to desired parts of the cell, an important aspect of crop design and biopharming.