Due to their endosymbiotic origin, chloroplasts harbor several subcompartments and membrane systems. Each of these has a different protein and lipid composition that dynamically changes either naturally during plant development or induced by environmental stimuli. Here, we describe a protocol for chloroplast envelope membrane subfractionation via discontinuous sucrose gradients, which offers the possibility to separate the different plastid subcompartments for several downstream applications. It is a strong tool for protein sublocalization studies as well as for tracking dynamic movement patterns. Furthermore, it can be combined with in vitro import studies of radioactively labeled proteins, which allows sublocalization of putative envelope proteins independent of the availability of specific antisera.
Keywords: Arabidopsis thaliana; Chloroplast isolation; Chloroplast subcompartments; Envelope membrane subfractionation; In vitro import; Pisum sativum; Protein dynamics.
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.