Defining the Core Proteome of the Chloroplast Envelope Membranes

Abstract

High-throughput protein localization studies require multiple strategies. Mass spectrometric analysis of defined cellular fractions is one of the complementary approaches to a diverse array of cell biological methods. In recent years, the protein content of different cellular (sub-)compartments was approached. Despite of all the efforts made, the analysis of membrane fractions remains difficult, in that the dissection of the proteomes of the envelope membranes of chloroplasts or mitochondria is often not reliable because sample purity is not always warranted. Moreover, proteomic studies are often restricted to single (model) species, and therefore limited in respect to differential individual evolution. In this study we analyzed the chloroplast envelope proteomes of different plant species, namely, the individual proteomes of inner and outer envelope (OE) membrane of Pisum sativum and the mixed envelope proteomes of Arabidopsis thaliana and Medicago sativa. The analysis of all three species yielded 341 identified proteins in total, 247 of them being unique. 39 proteins were genuine envelope proteins found in at least two species. Based on this and previous envelope studies we defined the core envelope proteome of chloroplasts. Comparing the general overlap of the available six independent studies (including ours) revealed only a number of 27 envelope proteins. Depending on the stringency of applied selection criteria we found 231 envelope proteins, while less stringent criteria increases this number to 649 putative envelope proteins. Based on the latter we provide a map of the outer and inner envelope core proteome, which includes many yet uncharacterized proteins predicted to be involved in transport, signaling, and response. Furthermore, a foundation for the functional characterization of yet unidentified functions of the inner and OE for further analyses is provided.

Assessment of the requirement for aquaporins in the thylakoid membrane of plant chloroplasts to sustain photosynthetic water oxidation

“…Although the plasma membrane marker H + -ATPase was also found in those preparations, a genuine chloroplast location for one or several isoforms of the PIP family was proposed. Most recently, TIP1;1 and PIP2;1 (also named PIP2A) were identified by mass spectrometry in the Arabidopsis envelope fraction [32].…”

Functions of plastid protein import and the ubiquitin–proteasome system in plastid development

“…Such autoubiquitination may lead to proteasomal degradation, as SP1 protein accumulated following proteasome inhibitor treatment [112]. This may explain why proteomic analyses of the chloroplast envelope have not uncovered SP1 [138,139]. Autoubiquitination is also likely to occur at SGR9, as it was shown that SGR9 protein can be stabilized in vivo by mutation of its RING domain causing loss of E3 ligase activity [132].…”

“…Interestingly, a recent proteomic study on purified chloroplast envelopes identified ubiquitin and some ubiquitin ligases [139]. However, contamination cannot be ruled out because of their high abundance.…”

Biogenesis of thylakoid membranes

“…Chloroplast outer and inner envelopes, as well as thylakoids, can be efficiently separated by centrifugation techniques, and their respective proteomes have been extensively characterized [28,[108][109][110]. These data revealed that lipid and carotenoid synthesis is localized to the envelope membranes.…”