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

作者：Daphné Seigneurin‐Berny, Norbert Rolland, Jérôme Garin, Jacques Joyard · 发表于：The Plant Journal · 年份：1999 · DOI：10.1046/j.1365-313x.1999.00511.x · 被引用次数：102 · 研究领域：Photosynthetic Processes and Mechanisms、Mass Spectrometry Techniques and Applications、Hemoglobin structure and function

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 true for membranes, such as plastid envelope membranes, that have a high lipid content, present a wide variety of specific functions and therefore contain a large number of unique, but minor, proteins. We have optimized a procedure, based on the differential solubilization of membrane proteins in chloroform/methanol mixtures, to extract and concentrate the most hydrophobic proteins from chloroplast envelope membrane preparations, while more hydrophilic proteins were excluded. In addition to previously characterized chloroplast envelope proteins, such as the phosphate/triose phosphate translocator, we have identified new proteins that were shown to contain putative transmembrane alpha-helices. Moreover, using different chloroform/methanol mixtures, we have obtained differential solubilization of envelope proteins as a function of their hydrophobicity. All the proteins identified were genuine chloroplast envelope proteins, most of them being localized within the inner membrane. Our procedure enables direct mapping (by classical SDS-PAGE) and identification of hydrophobic membrane proteins, whatever their isoelectric point was, that are minor components of specific subcellular compartments. Thus, it complements other techniques that give access to peripheral ...