Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins

Identification of rare hydrophobic membrane proteins is a major biological problem that is limited by the specific biochemical approaches required to extract these proteins from membranes and purify them. This is especially t rue for membranes, such as plastid envelope membranes, that have a high lip id content, present a wide variety of specific functions and therefore cont ain a large number of unique, but minor, proteins. We have optimized a proc edure, based on the differential solubilization of membrane proteins in chl oroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrop hilic proteins were excluded. In addition to previously characterized chlor oplast envelope proteins, such as the phosphate/triose phosphate translocat or, we have identified new proteins that were shown to contain putative tra nsmembrane alpha-helices. Moreover, using different chloroform/methanol mix tures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. Ail the proteins identified were genui ne chloroplast envelope proteins, most of them being localized within the i nner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoele ctric point was, that are minor components of specific subcellular compartm ents. Thus, it complements other techniques that give access to peripheral membrane proteins. If applied to various cell membranes, it is anticipated that it can expedite the identification of hydrophobic proteins involved in transport systems for ions or organic solutes, or it may act as signal rec eptors or to control metabolic processes and vesicle trafficking.