The function of the UBX-domain containing scaffold protein FAF1/PUX10 in lipid droplet degradation in seedlings and pollen tubes

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Lipid droplets are important storage organelles in plants, especially in pollen and seeds. The major proteins associated with these organelles are oleosins. Oleosins prevent lipid droplet fusion and their abundance determines lipid droplet size. During seed germination, oleosins are ubiquitinated and subsequently degraded. As this degradation is faster than the degradation of the oil itself, lipid droplet size increases. We identified a scaffold protein, NtFAF1/NtPUX10 at the lipid droplets of tobacco pollen tubes. Its Arabidopsis homologue, AtPUX10, was also targeted to lipid droplets, when transiently expressed in tobacco pollen tubes. From homologues in yeast in mammals, it is known that these proteins bind ubiquitinated proteins and recruit an AAA-type ATPase (Cdc48p in yeast) that can unfold proteins and remove them from membranes. As we could also show that the plant PUX10 proteins can recruit the Arabidopsis AtCDC48a to lipid droplets, we suspect that this whole complex could be involved in the removal of ubiquitinated oleosins from the lipid droplet. In two independent pux10 Arabidopsis mutant lines, we could show that lipid droplet size does not increase during seed germination, maybe because ubiquitinated oleosins are not removed, preventing the fusion of the lipid droplets. In order to further support our model we want to demonstrate that: 1. AtPUX10 localizes to seed lipid droplets by expressing it under the intrinsic promoter in Arabidopsis.2. AtPUX10 interacts directly with all three AtCDC48 isoforms via its UBX domain by performing transient BiFC-assays with full-length AtPUX10 and truncated versions in tobacco pollen tubes.3. AtPUX10 interacts with ubiquitin via its UBA-domain by Y2H-assay using the full-length AtPUX10 and truncated versions.4. The pux10-1 T-DNA insertion mutant phenotype (lipid droplets not increasing in size during germination) can be complemented by the full-length AtPUX10 but not by truncated versions missing either the UBA-domain or the UBX-domain.5. Oleosin degradation is slowed down in pux10-1 mutant seedlings by western blotting and tandem-mass-tag based quantification using LC-MS/MS.6. triacylglycerol degradation is affected in the pux10-1 mutant seedlings by measuring TAG-levels using GC-FID7. AtPUX10 is important for pollen tube growth by showing that pollen tube growth is slowed in vivo and that the transmission of the mutant allele via the pollen is reduced.All in all, the proposal aims for a better understanding of lipid droplet breakdown especially in the context of oleosin removal.