Finally, we isolated lipid bodies, micro somal and cytosolic protein fractions from tobacco proto plasts expressing oleosin GFP and carried out western blot analysis using an anti excellent validation GFP antibody. As shown in Fig. 4c, oleosin Inhibitors,Modulators,Libraries GFP was detected in the ER fraction, thus indi cating that, in our experimental conditions, LD are recov ered in such a fraction. A faint band of the molecular mass predicted for oleosin GFP was also found in the lipid body fraction at longer exposure. This observation supports the hypothesis that, in our experi mental conditions, LD are recovered mostly from the ER fraction. With the aim to better study the association of HPLF with LD, we carried out co expression of YFP tagged M. trunca tula HPLs and oleosin RFP chimeric constructs in tobacco protoplasts. As shown in Fig.

5b c, HPLF1 2 YFP chime ras showed a prevalent, even though not complete, co localisation Inhibitors,Modulators,Libraries with oleosin RFP fluorescence in LD. Co expression of OLE RFP and HPLF3 YFP chimeras did not succeed in targeting YPF to LD, which were only labelled by RFP. Finally, in tobacco protoplasts co expressing OLE RFP and HPLE1 YFP, YFP fluorescence was detected on the plastids as small spots similar to those reported in Fig. 2a and was physically separated by RFP fluorescence. However, in some cases LD, labelled by oleosin RFP, were very close to plastids and RFP and YFP fluorescences seemed to co localise. The physiological significance of such an association is currently unclear and further exper iments are in progress to clarify it.

To confirm the confocal microscopy results, we carried out sub cellular fractionation of tobacco protoplasts co expressing OLE RFP and HPLE F YFP. Plastidial, micro Inhibitors,Modulators,Libraries somal, lipid bodies and cytosolic protein fractions were isolated as described in the Materials section. As shown in Fig. 5e, the full chimera of HPLE1 YFP was detected only in the plastidial fraction. The lower molecular weight polypeptide immunodetected in the soluble protein sam ple may be due to some proteolytic degradation of the chi mera which produces a soluble polypeptide. Since no cytosolic distribution of fluorescence was observed in confocal images, it appeared evident that this fragment was unable to fold correctly and be fluorescent. HPLF1 YFP was mainly found in the cytosolic protein fraction, even though a clear band Inhibitors,Modulators,Libraries was also detected in the microsomal fraction, thus confirming the localisation of HPLF1 with ER associated LD.

A faint band was also detected in the plastid fraction. Inhibitors,Modulators,Libraries These results could be indicative of a limited interaction of HPLF with the outer membrane of this organelle. In this context, confocal images showed that, in some cases, YFP fluorescence was very close to plastids. Confocal worldwide distributors images also showed a nuclear localisation for HPLF YFP. This pattern was interpreted as a sign of solubility of the chimera in the cytosol.