![]() ![]() However, the function of most newly discovered lumenal proteins is not clear ( Kieselbach et al., 1998 Peltier et al., 2000). Lumenal proteins are involved in a number of well-characterized functions, such as water splitting, electron transport, and the violaxanthin cycle. A few small thylakoid proteins with a single transmembrane (TM) domain also have an lTP ( Thompson et al., 1998, 1999). In contrast, proteins located in the thylakoid lumen are targeted and translocated via a second transit peptide, the lumenal transit peptide (lTP), which is located directly C-terminal of the cTP. The inner envelope proteins, the peripheral thylakoid proteins located at the stromal site of the thylakoid membrane, and most of the integral thylakoid membrane proteins have no additional N-terminal transit peptides. ![]() Proteins then are directed into the inner envelope membrane, into the thylakoid membranes, or remain in the chloroplast stroma. After synthesis in the cytosol, the cTP is recognized at the chloroplast envelope and the precursor protein is translocated into the chloroplast, followed by processing of the cTP. This prediction is based on the presence of an N-terminal chloroplast transit peptide (cTP) in the nucleus-encoded chloroplast proteins (except for proteins localized in the chloroplast outer membrane) using the cellular localization program TargetP ( Emanuelsson et al., 2000). Plastids are predicted to contain between 10 to 15% of the nucleus-encoded gene products, corresponding to ∼2500 to 3500 proteins, indicating the importance of this organelle in the plant cell ( Arabidopsis Genome Initiative, 2000). Combined with rapidly expanding plant genome information, these technical improvements are expected to have a profound effect on plant biology (for discussion, see van Wijk, 2001).Ī significant subset of the Arabidopsis proteome is localized in different organelles (e.g., the mitochondria and different types of plastids). (1998), Pandey and Mann (2000), and Yates (2000) and references in the study by Blackstock and Mann (2000). Examples of excellent reviews on mass spectrometry and their application in biology include those by Jensen et al. ![]() The improved mass accuracy, mass resolution, and sensitivity of the latest generation of mass spectrometers allow the rapid identification of picomole to femtomole amounts of proteins and peptides. Importantly, the sequenced genome also makes it possible to take advantage of the dramatic improvements in biological mass spectrometry to study gene expression directly at the protein level and to determine protein localization and post-translational modifications efficiently in a systematic manner. The availability of the Arabidopsis genome now allows the classification of proteins according to predicted domain(s) and subcellular localization. Recently, the genome of the dicotyledon Arabidopsis was sequenced completely, and 25,498 genes were annotated ( Arabidopsis Genome Initiative, 2000). Many of the predicted lumenal proteins must be present at concentrations at least 10,000-fold lower than proteins of the photosynthetic apparatus. Thus, prime functions of the lumenal proteome include assistance in the folding and proteolysis of thylakoid proteins as well as protection against oxidative stress. ![]() Lumenal proteins with a typical twin-arginine translocation motif were predicted with good accuracy and sensitivity and included additional isomerases and proteases. Characteristics of the experimentally identified lumenal proteins and their orthologs were used for a genome-wide prediction of the lumenal proteome. These isomerases possibly are connected to a network of peripheral and lumenal proteins involved in antioxidative response, including peroxiredoxins, m-type thioredoxins, and a lumenal ascorbate peroxidase. Expression of five isomerases of different classes suggests strong (un)folding activity in the thylakoid lumen. Expression of a surprising number of paralogs was detected. Gene annotation of the identified proteins was corrected by experimental data, and an interesting case of alternative splicing was discovered. The identities of 81 proteins were established, and N termini were sequenced to validate localization prediction. Soluble thylakoid proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry. Experimental proteome analysis was combined with a genome-wide prediction screen to characterize the protein content of the thylakoid lumen of Arabidopsis chloroplasts. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |