Analysis of TLP40 in higher plants

3.7.1 Localisation of TLP40 within thylakoid membranes

Analysis of the protein sequence and import assay in vitro showed that TLP40 is indeed located in thylakoid lumen and can be either associated with the thylakoid membrane or exist in free form in the lumen (Fulgosi et al., 1998). In order to define more precisely the localisation of TLP40, a blue-native/SDS-PAGE analysis was performed to check whether TLP40 associated with some complex of thylakoid membranes. For the first dimension thylakoid membranes of spinach were solubilized with the detergent β-dodecyl-maltoside. The complexes were then separated by a 4 - 12% blue-native PAGE. Proteins constituting the complexes were separated in the second dimension by SDS-PAGE and transferred to a nitrocellulose membrane. The presence of TLP40 within thylakoid membrane and its association with photosynthetic complexes was tested immunologically with specific antibodies raised against TLP40, CP47 and cytochrome f.

HMW LMW CP47 TLP40 Cytf F ree pr ot ein s PSI I monome r PSI I dimer LHCII trimer Cy t b6 f comple x D1/ D 2/CP 47compl

Fig. 36. Western analysis of partially lysed spinach thylakoid membranes

separated by 2D gel electrophoresis. Proteins transferred into nitrocellulose

membrane were identified with antibodies raised against TLP40, cytochrome f and CP47. The direction of blue-native separation is indicated by the shortcut HMW, high molecular weight, and LMW, low molecular weight proteins. The major photosynthetic complexes are indicated on the first dimension according to

Western analysis of TLP40 after separation by 2D gel-electrophoresis showed two signals in the region of free proteins and in the region of the cytochrome b6f

complex. The two TLP40 signals differed in molecular weight and one could argue for possible TLP40 modifications (for example phosphorylation or other amino acid modifications) which alter the electrophoretic mobility of the protein.

3.7.2 Association of TLP40 with the cytochrome b6f complex in

tobacco

It was previously shown that TLP40 from spinach thylakoid membrane lysates separated by sucrose gradient partially comigrated with the cytochrome b6f

complex (Weber, 2001). Similar results were obtained when thylakoid membranes were separated by blue-native and SDS gel electrophoresis (see Fig. 36). Comigration of two proteins in a sucrose gradient or in a blue-native PAGE does not necessarily imply that proteins belong to the same complex. To check, whether TLP40 is associated with cytochrome b6f complex, the localisation of TLP40 was

analysed in tobacco plants deleted in petG (encoding subunit V) which resulted in a deficiency of the cytochrome b6f complex. Homoplastomic lines were able to

grow heterotrophically and demonstrated a pale green phenotype and a retarded growth rate under standard growth light (100 µE m-2s-1; Legen, 2003). Thylakoid membrane lysate of tobacco wild-type and mutant ∆petG were separated on a 0.1 – 1 M sucrose gradient. The gradient fractions were collected and loaded onto a SDS-PAG. Proteins were transferred onto a nitrocellulose membrane and tested with antibodies raised against cytochrome f and TLP40 (Fig.37).

As shown in Figure 37, TLP40 migrated mostly as a free protein and was not associated with the cytochrome b6f complex. This finding contrasts with what was

previously reported (Weber, 2001) A similar pattern was found when thylakoid membranes of mutant ∆petG were tested for the localisation of TLP40. Evidence that the absence of cytochrome b6f complex does not influence the migration

pattern of TLP40 indicated that TLP40 is not associated with cytochrome b6f

Cytf TLP40 TLP40 LHCI I Cyto chro me b6f com plex PSII m onom er 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 PSI m onom er 1 M 0.1 M

{

Fig. 37. Separation of thylakoid membranes from tobacco wild-type and

∆petG by sucrose gradient centrifugation. Thylakoid membranes were isolated

from fresh harvested leaves and solubilized with β−dodecyl-maltoside. Solubilized membranes were loaded onto a linear 0.1 – 1 M sucrose gradient. Photosynthetic complexes were separated by ultracentrifugation for 16 h at 4°C. The gradient was collected and protein fractions were loaded on a 12.5% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and tested with antibodies raised against TLP40 and cytochrome f. The fractions of tobacco wild-type and ∆petG were kindly provided by Dr. Julia Legen.

3.7.3 Association of TLP40 with thylakoid membranes in vivo

It was suggested that TLP40 in chloroplasts could be involved in the regulation of the phosphatase that dephosphorylates PSII subunits (Fulgosi et al., 1998; Vener

et al., 1999; Rokka et al., 2000). In particular, it was proposed for TLP40 to be a negative regulator of protein phosphatase: upon binding of TLP40 to thylakoid membranes and thus to phosphatase, its activity could be inhibited. The activation of phosphatase coincides with the release of TLP40 into the lumen (Vener et al., 1999; Rokka et al., 2000). This hypothesis was tested in vitro by detecting the association/dissociation of TLP40 with thylakoid membranes under different temperature regimes. It was observed that the rise of temperature coincided with an increase of the dephosphorylation activity and with a release of TLP40 into the lumen (Rokka et al., 2000). The association/dissociation of TLP40 to the lumen

Afterwards, the plants were adapted to different light and temperature regimes for different periods. Thylakoids were extracted from acclimated plants and separated into lumenal and thylakoid membrane proteins. Fractions were loaded onto SDS- PAGE, transferred to a nitrocellulose membrane and tested with polyclonal antibodies raised against TLP40 (Fig. 38).

Chl Thl Lum Chl Thl Lum Chl Thl Lum Chl Thl Lum

NC HL HT LT

PsbO TLP40

Fig. 38. Association of TLP40 with thylakoid membranes. Spinach plants were

grown for 5 weeks in a green house (normal conditions, NC, 25°C). Later on, plants were adapted to HL (750 µE m-2s-1, 25°C), high temperature (HT, 42°C, 50 µE m-2 s-1) for 2 h and to low temperature (LT, 4°C, 50 µE m2 s-1) for 24 h. Thylakoid membranes were extracted and separated into lumenal and thylakoid membrane proteins. For each experiment chloroplast (chl), thylakoid (thl) and lumenal (lum) fractions were separated on a 12.5% SDS-PAGE, transferred to a nitrocellulose membrane and tested with antibodies raised against TLP40. PsbO protein was used as a control for lumenal proteins.

Figure 38 shows that the adaptation of spinach plants to high temperature did not increase the release of TLP40 into the lumenal compartment as it was suggested previously from experiments in vitro (Rokka et al., 2000). In addition, it was checked whether other conditions, could cause the dissociation of TLP40 from thylakoid membrane. Spinach plants were acclimated to low temperature and to high light, conditions that normally impair the function of photosynthetic complexes. Similar to high light TLP40 was mostly found attached to the membrane. These results are controversial under our chosen conditions with those reported by Rokka et al., (2000).