Studying Arabidopsis envelope protein localization and topology using thermolysin and trypsin proteases

Chloroplasts are metabolically important organelles that perform many essential functions within plant cells. The chloroplasts can be subdivided into six distinct sub-compartments to which a protein may be ultimately targeted. These sub-compartments are defined as the outer envelope membrane (OEM), the inner envelope membrane (IEM), the thylakoid membrane, and three aqueous sub-compartments - the intermembrane space (IMS), the stroma, and the thylakoid lumen. The process by which proteins are targeted to the chloroplastic envelope membrane remains a challenging question in cell biology. Our understanding of protein targeting to the OEM is very limited, whereas targeting of membrane proteins to the IEM appears to utilize at least two targeting pathways called the stop-transfer and the conservative sorting (or post-import) pathways. Furthermore, once a membrane protein arrives at the envelope membrane, our understanding of how it achieves its final topology remains limited. One method that can be used to determine the topology of an envelope membrane protein is to apply the "dual protease" strategy. This approach involves several steps: first, performing an in vitro import assay; second, applying a "dual protease" protection assay using thermolysin and trypsin; and finally, isolating and analyzing chloroplastic subcellular fractionations (i.e., total membrane and soluble fractions). By using this multistep approach, one can gain critical information regarding the final topology of an OEM or IEM protein. Likewise, the "dual protease" approach may help in elucidating the possible targeting pathway that a membrane protein utilizes prior to its insertion into the envelope membrane.