Tandem mass spectrometry identifies sites of three post-translational modifications of spinach light-harvesting chlorophyll protein II. Proteolytic cleavage, acetylation, and phosphorylation.
The photosynthetic membranes of spinach (Spinacia oleracea L.) chloroplasts were incubated with [gamma-32P] ATP. When the thylakoid membrane kinase was activated with light, the 25- and 27-kDa forms of the light-harvesting chlorophyll a/b protein (LHC II) were phosphorylated on their amino termini. Treatment of the membranes with proteinase K or thermolysin released phosphopeptides which were purified by ferric ion affinity chromatography and reverse phase high performance liquid chromatography. Sequencing of the phosphopeptides was performed with tandem quadrupole mass spectrometry. Three different phosphopeptides Ac-RKTAGKPKT, Ac-RKTAGKPKN, and Ac-RKSAGKPKN originating from class I LHC II were examined after release by thermolysin. One phosphopeptide, Ac-RRTVKSAPQ, originating from class II LHC II was examined after release by proteinase K. Each of the four LHC II phosphopeptides was derived from the amino terminus of a distinct protein. Peptides were acetylated at their amino-terminal arginine and were phosphorylated on either threonine or serine in the third position. We conclude that proteolytic processing of pre-LHC II occurs at a conserved methionyl-arginyl bond and is followed by amino-terminal acetylation of the arginine and nearby phosphorylation of the mature LHC II. Eight different peptides were synthesized in acetylated and nonacetylated forms as substrates for the thylakoid membrane kinase. From a comparison of the kinetics of phosphate incorporation into the peptides, we conclude that basic residues on both sides of the phosphorylation site are important for enzyme recognition. Acetylation of the amino terminus is not required for phosphorylation.
Michel
H
H
Biology Department, Brookhaven National Laboratory, Upton, New York 11973.
Griffin
P R
PR
Shabanowitz
J
J
Hunt
D F
DF
Bennett
J
J
D000107:Acetylation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D045342:Light-Harvesting Protein Complexes; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D000596:Amino Acids; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins