Light-harvesting chlorophyll a/b-binding protein inserted into isolated thylakoids binds pigments and is assembled into trimeric light-harvesting complex.
The light-harvesting chlorophyll a/b-binding protein (LHCP) is largely protected against protease (except for about 1 kD on the N terminus) in the thylakoid membrane; this protease resistance is often used to assay successful insertion of LHCP into isolated thylakoids in vitro. In this paper we show that this protease resistance is exhibited by trimeric light-harvesting complex of photosystem II (LHCII) but not by monomeric LHCII in which about 5 kD on the N terminus of LHCP are cleaved off by protease. When a mutant version of LHCP that is unable to trimerize in an in vitro reconstitution assay is inserted into isolated thylakoids, it gives rise to only the shorter protease digestion product indicative of monomeric LHCII. We conclude that more of the N-terminal domain of LHCP is shielded in trimeric than in monomeric LHCII and that this difference in protease sensitivity can be used to distinguish between LHCP assembled in LHCII monomers or trimers. The data presented prove that upon insertion of LHCP into isolated thylakoids at least part of the protein spontaneously binds pigments to form LHCII, which then is assembled in trimers. The dependence of the protease sensitivity of thylakoid-inserted LHCP on the oligomerization state of the newly formed LHCII justifies caution when using a protease assay to verify successful insertion of LHCP into the membrane.
Botanisches Institut III, Universität, München, Germany.
A
A
Kuttkat
R
R
Grimm
H
H
Paulsen
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008027:Light; D045342:Light-Harvesting Protein Complexes; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015388:Organelles / Q000378:metabolism*; D018532:Pisum sativum / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D045342:Light-Harvesting Protein Complexes; D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011498:Protein Precursors; D011994:Recombinant Proteins