PLANT PHYSIOLOGY , Vol 109, Issue 4 1267-1276, Copyright © 1995 by American Society of Plant Biologists
|
BIOCHEMISTRY AND ENZYMOLOGY |
Light-Harvesting Chlorophyll a/b-Binding Protein Inserted into Isolated Thylakoids Binds Pigments and Is Assembled into Trimeric Light-Harvesting Complex
A. Kuttkat, R. Grimm and H. Paulsen
Botanisches Institut III der Universitat, Menzinger Strasse 67, D-80638 Munchen, Germany (A.K., H.P.)
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.
