Transit Peptide Cleavage Sites of Integral Thylakoid Membrane Proteins

Abstract

A set of 58 nuclearly encoded thylakoid-integral membrane proteins from four plant species was identified, and their amino termini were assigned unequivocally based upon mass spectrometry of intact proteins and peptide fragments. The dataset was used to challenge the Web tools ChloroP, TargetP, SignalP, PSORT, Predotar, and MitoProt II for predicting organelle targeting and transit peptide proteolysis sites. ChloroP and TargetP reliably predicted chloroplast targeting but only reliably predicted transit peptide cleavage sites for soluble proteins targeted to the stroma. SignalP (eukaryote settings) accurately predicted the transit peptide cleavage site for soluble proteins targeted to the lumen. SignalP (Gramnegative bacteria settings) reliably predicted peptide cleavage of integral thylakoid proteins inserted into the membrane via the "spontaneous" pathway. The processing sites of more common thylakoid-integral proteins inserted by the signal recognition peptide-dependent pathway were not well predicted by any of the programs. The results suggest the presence of a second thylakoid processing protease that recognizes the transit peptide of integral proteins inserted via the spontaneous mechanism and that this mechanism may be related to the secretory mechanism of Gram-negative bacteria. Molecular & Cellular Proteomics 2:1068 -1085, 2003.Advances in genome sequencing (1-3) have provided a huge resource that is driving the field of bioinformatics. Besides providing a readout that approximates the primary structure of a particular gene product, there are many facets of expression, such as organellar targeting, that can be addressed via computational analysis of genomic data. Posttranslational modification sites, including signal peptide cleavage sites, may be predicted, although the accuracy of such predictive algorithms is often rooted in the knowledge base used for their development.Proteomics, the systematic study of large sets of proteins expressed by a particular cell type or tissue, is largely possible because of recent technological and computational advances. Contemporary proteomics is based on the following three components: analytical separation of proteins from complex starting material, identification of the proteins, and subsequent sorting and classification of the datasets using bioinformatics software tools. The availability of complete genome sequences, improvements in techniques for protein separation and displays (4 -6), and new developments in mass spectrometry (MS) 1 for protein identification (7-10) have arguably outpaced developments in bioinformatics. Using the complete mitochondrial, plastid, and nuclear Arabidopsis thaliana genomes (2, 11, 12) and results from recent proteomics experiments (13-16), it is possible to test the performance of algorithms that are commonly used to predict organellar targeting of nuclear gene products and transit peptide cleavage sites. Thus, it is timely to reevaluate the various tools at our disposal. The chloroplast is a fascinating organelle not only because ...

Environmentally Modulated Phosphoproteome of Photosynthetic Membranes in the Green Alga Chlamydomonas reinhardtii

“…This implies that many of the nuclear encoded proteins are processed by the algal chloroplast machinery differently from the "conventional" mechanistic rules used to design the prediction programs for chloroplast targeting and the length of cleavable transit peptides (68,75). A similar conclusion has also been drawn after characterization of mature thylakoid-integral proteins from plants (67). The successful identification of all these in vivo post-translational modifications is credited to the experimental approach of vectorial proteomics (76,77) used in this study.…”

“…5. Similarly, the experimentally determined amino termini of LHCII proteins from four plant species also differed from those allocated by the prediction programs (67). The first 15 amino acids in all four mature major LHCII proteins from Chlamydomonas are identical and contain the phosphorylated threonine residues surrounded by basic amino acids (see Fig.…”

“…6C and Table II). Prediction of transit peptides in plant LHCII proteins by the ChloroP method was found inaccurate as well (67). The earlier attempts of CP26 amino-terminal sequencing by Edman degradation failed due to natural blocking of its amino terminus (69).…”

Comparative Large Scale Characterization of Plant versus Mammal Proteins Reveals Similar and Idiosyncratic N-α-Acetylation Features

“…Although chloroplast imported proteins have been previously described to be acetylated after the cleavage of the cTP (38,42,47), our current study gives for the first time an idea of the extent of this modification in this organelle with more than 220 characterized proteins. Although some previous studies (38,42) suggest that the NAA is a post-translational modification on a limited number of chloroplast imported protein, the present results clearly highlight the high occurrence of this modification in the chloroplast compartment.…”

“…Because such NAA occurs at the neo-N terminus uncovered after the cleavage of the chloroplast transit peptide (cTP), this modification is clearly post-translational. Although a few similar cases have been described before (38,42,(47)(48)(49), NAA of chloroplast proteins appears to be a widespread modification in this organelle that has never been described before to this extent. Substrate specificity appears to be close to the cytosolic NatA complex, suggesting a dedicated Nat occurring in this organelle.…”