Date Published: November 7, 2017
Publisher: Public Library of Science
Author(s): Nuria Andrés-Colás, Dominique Van Der Straeten, Miguel A. Blazquez.
Pentatricopeptide repeat proteins are one of the major protein families in flowering plants, containing around 450 members. They participate in RNA editing and are related to plant growth, development and reproduction, as well as to responses to ABA and abiotic stresses. Their characteristics have been described in silico; however, relatively little is known about their biochemical properties. Different types of PPR proteins, with different tasks in RNA editing, have been suggested to interact in an editosome to complete RNA editing. Other non-PPR editing factors, such as the multiple organellar RNA editing factors and the organelle RNA recognition motif-containing protein family, for example, have also been described in plants. However, while evidence on protein interactions between non-PPR RNA editing proteins is accumulating, very few PPR protein interactions have been reported; possibly due to their high instability. In this manuscript, we aimed to optimize the conditions for non-denaturing protein extraction of PPR proteins allowing in vivo protein analyses, such as interaction assays by co-immunoprecipitation. The unusually high protein degradation rate, the aggregation properties and the high pI, as well as the ATP-dependence of some PPR proteins, are key aspects to be considered when extracting PPR proteins in a non-denatured state. During extraction of PPR proteins, the use of proteasome and phosphatase inhibitors is critical. The use of the ATP-cofactor reduces considerably the degradation of PPR proteins. A short centrifugation step to discard cell debris is essential to avoid PPR precipitation; while in some cases, addition of a reductant is needed, probably caused by the pI/pH context. This work provides an easy and rapid optimized non-denaturing total protein extraction protocol from plant tissue, suitable for polypeptides of the PPR family.
Pentatricopeptide repeat containing proteins (PPRs) are found in some prokaryotes and almost all eukaryotes. In plants, they represent one of the largest protein families . They play a major role in RNA metabolism [2, 3]. PPR proteins are essential in plant reproduction, where their absence often causes lethality [4–7]. In addition, they have been related to plant growth and development, through regulation of energy metabolism and responses to ABA, as well as to abiotic stresses [1, 8]. PPR proteins are also associated with photosynthetic defects, aberrant leaf development, changes in leaf pigmentation, tolerance to inhibitors of different biosynthetic pathways, and restoration of pollen fertility . Most of the plant PPR proteins are located in mitochondria (65%) or chloroplasts (17%) . PPR proteins are named based on the presence of around 35-amino-acid motifs, repeated in tandem . Depending on the extra motifs, the PPR proteins are classified in different subfamilies (P-type and PLS) and subgroups (PLS, E, E+ and DYW). Each type of PPR protein is suggested to play a different task in RNA editing and to interact in modular editosomes for a complete editing event [10–12]. Besides in silico prediction of their characteristics , relatively little is known about their biochemical properties. Therefore, the working mechanisms of PPR proteins are by far not completely understood.
The standard protein extraction buffer used for PPR proteins in this paper was supplemented with an EDTA-free protease inhibitor cocktail, which lacks any specific inhibitor of metalloproteases. Given that some RNA editing factors have been described to require divalent cations, which are chelated by EDTA, the extraction buffer was kept free of EDTA to avoid the non-functionality of the PPR proteins that could possibly interfere with their solubility.
The high protein degradation rate, the aggregation properties, the need of ATP as a co-factor, and the high pI are key aspects to be considered when extracting PPR proteins in a non-denatured state. Therefore, the use of specific proteasome and phosphatase inhibitors is a critical point when extracting PPR proteins. ATP is also needed to avoid protein degradation once the PPR proteins have been extracted. Short centrifugation steps are another essential element to avoid PPR precipitation. Last but not least, in some cases, a reductant such as DTT or β-mercaptoethanol can be needed to prevent PPR protein precipitation, presumably caused by the pI/pH context.