The Mobile Thylakoid Phosphoprotein TSP9 Interacts with the Light-harvesting Complex II and the Peripheries of Both Photosystems

Maria Hansson,Tiphaine Dupuis,Ragna Strömquist,Bertil Andersson,Alexander V Vener,Inger Carlberg

doi:10.1074/jbc.m605833200

Maria Hansson, Tiphaine Dupuis + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m605833200

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Abstract

The localization of the plant-specific thylakoid-soluble phosphoprotein of 9 kDa, TSP9, within the chloroplast thylakoid membrane of spinach has been established by the combined use of fractionation, immunoblotting, cross-linking, and mass spectrometry. TSP9 was found to be exclusively confined to the thylakoid membranes, where it is enriched in the stacked grana membrane domains. After mild solubilization of the membranes, TSP9 migrated together with the major light-harvesting antenna (LHCII) of photosystem II (PSII) and with PSII-LHCII supercomplexes upon separation of the protein complexes by either native gel electrophoresis or sucrose gradient centrifugation. Studies with a cleavable cross-linking agent revealed the interaction of TSP9 with both major and minor LHCII proteins as identified by mass spectrometric sequencing. Cross-linked complexes that in addition to TSP9 contain the peripheral PSII subunits CP29, CP26, and PsbS, which form the interface between LHCII and the PSII core, were found. Our observations also clearly suggest an interaction of TSP9 with photosystem I (PSI) as shown by both immunodetection and mass spectrometry. Sequencing identified the peripheral PSI subunits PsaL, PsaF, and PsaE, originating from cross-linked protein complexes of around 30 kDa that also contained TSP9. The distribution of TSP9 among the cross-linked forms was found to be sensitive to conditions such as light exposure. An association of TSP9 with LHCII as well as the peripheries of the photosystems suggests its involvement in regulation of photosynthetic light harvesting.

Highlights

Different kinases [2,3,4,5] as well as several phosphatases (6 – 8) are believed to participate
Most proteins undergoing protein phosphorylation in the thylakoid membrane are associated with photosystem II (PSII)2 and its light-harvesting antenna (LHCII) [9, 10]
This type of fractionation does not exclude the presence of minor amounts of TSP9 elsewhere yet clearly shows that TSP9 is associated with the thylakoid membrane in dark-adapted plants

Summary

EXPERIMENTAL PROCEDURES

Preparation and Subfractionation of Thylakoid Membranes— Thylakoid membranes were prepared from 7-week-old darkadapted spinach as described [22] and resuspended in 0.1 M sorbitol, 25 mM Tricine, pH 7.9, 5 mM MgCl2 and 10 mM KCl. After centrifugation at 14,000 ϫ g for 15 min, the supernatant was supplemented with 0.1 volume sample buffer (100 mM BisTris-HCl, pH 7.0, 0.5 M ⑀-amino-n-caproic acid, 30% (w/v) sucrose, 50 mg/ml Serva blue G) and subjected to BN-PAGE with a gradient of 5–13.5% acrylamide in the separation gel. For separation of proteins in the second dimension, lanes were cut out and incubated with 5% ␤-mercaptoethanol in SDS sample buffer for 30 min at room temperature and subjected to SDS-PAGE with 15% acrylamide in the separation gel in the second dimension. Sucrose Gradient Centrifugation—Thylakoid membranes were resuspended in 25 mM Tricine, pH 7.9, 5 mM MgCl2, 10 mM KCl to a concentration of 1 mg chl/ml and solubilized with 1% n-dodecyl-␤-D-maltoside on ice for 10 min. The collision-induced dissociation of selected precursor ions was performed using the instrument settings recommended by Applied Biosystems and manual control of collision energy

RESULTS

Homolog proteinc kDa

DISCUSSION