Isolation of pigment-binding early light-inducible proteins from pea.

The early light-inducible proteins (ELIPs) in chloroplasts possess a high sequence homology with the chlorophyll a/b-binding proteins but differ from those proteins by their substoichiometric and transient appearance. In the present study ELIPs of pea were isolated by a two-step purification strategy: perfusion chromatography in combination with preparative isoelectric focussing. Two heterogeneous populations of ELIPs were obtained after chromatographic separation of solubilized thylakoid membranes using a weak anion exchange column. One of these populations contained ELIPs in a free form providing the first isolation of these proteins. To prove whether the isolated and pure forms of ELIP bind pigments, spectroscopic and chromatographic analysis were performed. Absorption spectra and TLC revealed the presence of chlorophyll a and lutein. Measurements of steady-state fluorescence emission spectra at 77 K exhibited a major peak at 674 nm typical for chlorophyll a bound to the protein matrix. The action spectrum of the fluorescence emission measured at 674 nm showed several peaks originating mainly from chlorophyll a. It is proposed that ELIPs are transient chlorophyll-binding proteins not involved in light-harvesting but functioning as scavengers for chlorophyll molecules during turnover of pigment-binding proteins.

Synthesis and assembly of the D1 protein into photosystem II: processing of the C-terminus and identification of the initial assembly partners and complexes during photosystem II repair.

In previous studies [van Wijk, K. J., Bingsmark, S., Aro, E.-M., & Andersson, B. (1995) J. Biol. Chem. 270, 25685-25695; van Wijk, K. J., Andersson, B., & Aro, E.-M. (1996) J. Biol. Chem 271, 9627-9636], we have demonstrated that D1 protein synthesized in isolated chloroplasts and thylakoids is incorporated into the photosystem II (PSII) core complex. By pulse-chase experiments in these in vitro systems, followed by sucrose gradient fractionation of solubilized thylakoid membranes, it was shown that this assembly proceeded stepwise; first the D1 protein was incorporated to form a PSII reaction center complex (PSII rc), and through additional assembly steps the PSII core complex was formed. In this study, we have analyzed this assembly process in more detail, with special emphasis on the initial events, through further purification and analysis of the assembly intermediates by nondenaturing Deriphat-PAGE and by flatbed isoelectric focusing. The D2 protein was found to be the dominant PSII reaction center protein initially associating with the new D1 protein. This strongly suggests that the D2 protein is the primary "receptor" or stabilizing component during or directly after synthesis of the D1 protein. After formation of the D1-D2 heterodimer, cyt b559 became attached, whereas the psbI gene product was assembled as a subsequent step, thereby forming a PSII reaction center complex. Subsequent formation of the PSII core occurred by binding of CP47 and then CP43 to the PSII rc. The rapid radiolabeling of a minor population of a PSII core subcomplex without CP43 indicated that an association of newly synthesized D1 protein with a preexisting complex consisting of D2/cyt b55q/psbI gene product/CP47 was possibly occurring, in parallel to the predominant sequential assembly pathway. The kinetics of synthesis and processing of the precursor D1 protein were followed in isolated chloroplasts and were compared with its incorporation into PSII assembly intermediates. No precursor D1 protein was found in PSII core complexes, indicating either that incorporation into the PSII core complex facilitates the cleavage of the C-terminus or, more likely, that processing is more rapid than the assembly into the PSII core.

Isolation of hydrophobic membrane proteins by perfusion chromatography--purification of photosystem II reaction centers from spinach chloroplasts.

Ion exchange perfusion chromatography has been introduced for the isolation of hydrophobic membrane protein complexes from thylakoid membranes of spinach chloroplasts. By using this chromatographic technique, previously shown to be useful for the rapid isolation of soluble proteins (Regnier, F. E. (1991) Nature 350, 634-635), we have been able to isolate oxygen evolving photosystem II core complexes and photosystem II reaction center particles. Pure reaction centers could be isolated from photosystem II core complexes after a chromatographic step requiring only 6.5 mm, which is a substantial improvement in comparisons with previous procedures. The entire preparation of photosystem II core complexes and reaction center II particles could be completed in less than 2 h. The use of perfusion chromatography, as a versatile method for the isolation of hydrophobic membrane proteins from photosynthetic membranes, as well as for other biological membranes, will be discussed.

Degradation of the light-stress protein is mediated by an ATP-independent, serine-type protease under low-light conditions.

Green plants respond to light stress by induction of the light-stress proteins (ELIPs). These proteins are stable as long as the light stress persists but are very rapidly degraded during subsequent low light conditions. Here we report that the degradation of ELIPs is mediated by an extrinsic, thylakoid-associated protease which is already present in the membranes during light stress conditions. Partial purification of the protease by perfusion chromatography indicates that this proteolytic activity may be represented by a protein with an apparent molecular mass of 65 kDa. The ELIP-directed protease is localized in the stroma lamellae of the thylakoid membranes and does not require ATP or additional stromal factors for proteolysis. The protease has an optimum activity at pH 7.5-9.5 and requires Mg2+ for its activity. The ELIP-degrading protease show an unusual temperature sensitivity and becomes reversibly inactivated at temperatures below 20 degree C and above 30 degree C. Studies with protease inhibitors indicate that this enzyme belongs to the serine class of proteases. The enhanced degradation of ELIP in isolated thylakoid membranes after addition of the ionophore nigericin suggests that a trans-thylakoid delta pH or changes in ionic strength may be involved in the mechanism of protease activation.

Rapid isolation of photosystem I chlorophyll-binding proteins by anion exchange perfusion chromatography.

With the new method of anion exchange perfusion chromatography we have devised an extremely rapid technique to subfractionate spinach Photosystem I into its chlorophyll a containing core complex and various components of the Photosystem I light-harvesting antenna (LHC I). The isolation time for the LHC I subcomplexes following solubilisation of native Photosystem I was reduced from 50 h using traditional density centrifugation procedures down to only 10-25 min by perfusion chromatography. Within this very short period of isolation, LHC I has been obtained as subfractions highly enriched in Lhca2+3 (LHC I-680) and Lhca1+4 (LHC I-730). Moreover, other highly enriched subfractions of LHC I such as Lhca2, Lhca3 and Lhca1+2+4 were obtained where the later two populations have not previously been obtained in a soluble form and without the use of SDS. These various subfractions of the LHC I antenna have been characterised by absorption spectroscopy, 77 K fluorescence-spectroscopy and SDS-PAGE demonstrating their identities, functional intactness and purity. Furthermore, the analyses located a chlorophyll b pool to preferentially transfer its excitation energy to the low energy F735 chromophore, and located specifically the origin of the 730 nm fluorescence to the Lhca4 component. It was also revealed that Lhca2 and Lhca3 have identical light-harvesting properties. The isolated Photosystem I core complex showed high electron transport capacity (1535 µmoles O2 mg Chl(-1) h(-1)) and low fluorescence yield (0.4%) demonstrating its high functional integrity. The very rapid isolation procedure based upon perfusion chromatography should in a significant way facilitate the subfractionation of Photosystem I proteins and thereby allow more accurate functional and structural studies of individual components.

Perfusion chromatography-a new procedure for very rapid isolation of integral photosynthetic membrane proteins.

The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states.