Topology of PPP4 and TMP14

As mentioned afore, TMP14 was first described, because of its posttranslational modification by phosphorylation of the N-terminus (Hansson and Vener, 2003), which was found by trypsin digestion of all stromal side exposed proteins and subsequent enrichment of phosphopeptides. Because TMP14 has two predicted transmembrane domains, this indicates that both termini are stromal exposed. To determine PPP4 and to verify TMP14 topology, thylakoids were subjected to mild digestion with trypsin for 10 min with the aim to solely target tryptic sites on the stromal side of the thylakoid membrane. Both proteins contain multiple trypsin cleavage sites in their N- terminus, which also bears the sequence of the epitopes, against which specific antibodies were made. Therefore, stromal side treatment with trypsin of a topology in which the N- and C-termini of

the proteins were exposed to the stromal side (topology 1, Figure 3.1.14B), would result in

destruction of the epitopes and absence of detectability. Because PPP4 has one tryptic site in the transmembane regions connecting loop, treatment with trypsin on a stromal exposed loop topology (topology 2) would result in detectable product of 5 kDa for PPP4 with the specific antibody. The tryptic digest was additionally performed with thylakoids of tmp14 and ppp4 mutants in order to

evaluate if the absence of one interaction partner would change accessibility of the other to the protease, and with PPP4:c-myc expressing plants as a confirmation for PPP4 topology. Because the

TMP14 antibody reacts unspecifically with chlorophyll on the PVDF membrane, thylakoid proteins were precipitated with acetone after the trypsin treatment to avoid this unspecific interaction during Western detection. The reaction of the antibody with the low molecular chlorophyll could interfere with the detection of smaller TMP14 degradation products. As an indication that tryptic proteolysis took place only at the stromal side of the membrane and no unspecific cleavage of lumenal proteins occurred, Western analysis of the treated thylakoid extracts was performed with an antibody against the lumenal protein PsbO, a component of the PSII oxygen evolving complex (Figure 3.1.14A).

This shows that after 10 min of digestion PsbO remains intact. Hybridisation with an antibody against the stromal side localised AtpB confirms activity of trypsin. Western detection with the specific antibodies against TMP14 and PPP4 clearly indicates that both proteins are protected from trypsin treatment, because their amount does not decrease, but even seems to increase after the protease treatment. This result could be due to a negative effect of associated proteins on the accessibility of PPP4 and TMP14 to the detergent SDS in the loading buffer after acetone precipitation. If the associated proteins, which together with PPP4 and TMP14 precipitate in acetone as a partly insoluble fraction, are partially removed by proteolytic cleavage before the precipitation, higher amounts of PPP4 and TMP14 would be soluble. A 10 min trypsin treatment of TMP14 results in a detectable fragment of smaller size, which indicates that the TMP14 N-terminus with its 8 tryptic sites is heavily protected and cleavage might have occurred at an arginine residue two amino acids upstream of the predicted transmembrane domain or at the only lumenal exposed tryptic site. Digested PPP4 exhibits one degradation product after 10 min of slightly smaller size. Therefore topology 1 with stromal localised N- and C-terminus appears likely. Verification of this result arises from PPP4:c-myc digestion. The amount of detectable c-myc is decreasing and an increasing amount of normal sized PPP4 indicates that the c-myc tag is cleaved off.

Fig 3.1.14 Determination of PPP4 topology by stromal side tryptic digest of thylakoid proteins

(A) Thylakoids were isolated from WT, 35S::PPP4:c-myc, ppp4 and tmp14 and incubated with 10 µg/ml trypsin.

Samples were taken at 0, 10 and 20 min after the addition of trypsin and proteins were separated by SDS-PAGE. Immuno-detection was carried out with specific antibodies against AtpB, as a control for trypsin activity, PsbO as a control for absence of trypsin in the lumen, TMP14, PPP4 and c-myc. Asterisks indicate degradation products. (B)

Schematic drawings of probable topologies. Scissors indicate possible luminal accessible tryptic sites. Red boxes indicate location of epitopes used for antibody production. Dashed green line stands for c-myc tag fused to PPP4. For PPP4 topology 1 could be supported.

Additionally, lack of PPP4 does not result in enhanced degradation of TMP14 or vice versa. Both proteins even seem to be more protected in the absence of the interaction partner, as no degradation product can be detected in the respective other mutant. Interestingly, the increase by trypsin treatment of Western detectable TMP14 is even higher in the ppp4 mutant background as compared

speculatively because of an increased association with other hydrophobic proteins. Two results obtained by this experiment suggest that TMP14 and PPP4 are associated with other proteins in the thylakoid membrane: (i) hardly any digestion of PPP4 and TMP14 takes place despite the high number of tryptic sites in the experimentally shown stromal localised N- and C- termini (TMP14: 8 N-terminal, 3 C-terminal; PPP4: 3 N-terminal, 3 C-terminal). (ii) PPP4 and TMP14 from untreated thylakoids seem to only be partly solubilised in Laemmli buffer, whereas solubilisation is markedly increased if samples are treated with trypsin.

3.1.10 Analysis of thylakoid protein composition