Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins.
The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.
Peltier
J B
JB
Department of Biochemistry, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden.
Friso
G
G
Kalume
D E
DE
Roepstorff
P
P
Nilsson
F
F
Adamska
I
I
van Wijk
K J
KJ
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism*; D016208:Databases, Factual; D004579:Electron Transport; D015180:Electrophoresis, Gel, Two-Dimensional; D020224:Expressed Sequence Tags; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D018506:Gene Expression Regulation, Plant; D015151:Immunoblotting; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000737:chemistry / Q000235:genetics; D045322:Photosynthetic Reaction Center Complex Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D010940:Plant Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D020539:Sequence Analysis, Protein; D012984:Software; D020524:Thylakoids / Q000378:metabolism
D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins