Transit Peptide Cleavage Sites of Integral Thylakoid Membrane Proteins*

A set of 58 nuclearly encoded thylakoid-integral mem-brane 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 tar-geted to the stroma. SignalP (eukaryote settings) accu-rately predicted the transit peptide cleavage site for sol-uble proteins targeted to the lumen. SignalP (Gram-negative bacteria settings) reliably predicted peptide cleavage of integral thylakoid proteins inserted into th