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

脂滴是植物中重要的贮藏细胞器，尤其是在花粉和种子中。与这些细胞器相关的主要蛋白质是油球蛋白。石油素阻止脂滴融合，其丰度决定了脂滴的大小。在种子萌发过程中，油球蛋白被泛素化，随后被降解。由于这种降解比油本身的降解更快，所以脂滴的尺寸会增加。我们在烟草花粉管的脂滴中鉴定出一个支架蛋白NtFAF1/NtPUX10。它的拟南芥同源基因AtPUX10在烟草花粉管中瞬时表达时，也针对脂滴。从哺乳动物酵母中的同源物中，我们知道这些蛋白质结合泛素化的蛋白质，并招募一种AAA型ATPase(酵母中的CDC48P)，该酶可以解开蛋白质并将它们从膜上移除。由于我们还可以证明植物PUX10蛋白可以将拟南芥AtCDC48a招募到脂滴中，我们怀疑整个复合体可能参与了从脂滴中去除泛素化的油球蛋白。在两个独立的pux10拟南芥突变体系中，我们可以证明在种子萌发过程中脂滴大小没有增加，这可能是因为泛素化的油球蛋白没有被去除，阻止了脂滴的融合。为了进一步支持我们的模型，我们想要证明：1.AtPUX10通过在拟南芥的内在启动子下表达，定位于种子脂滴。AtPUX10通过与烟草花粉管中的全长AtPUX10和截短版本的AtPUX10进行瞬时BIFC分析，通过其Ubx结构域直接与所有三种AtCDC48亚型相互作用。通过使用全长AtPUX10和截短版本的Y2H-分析，AtPUX10通过其UBA结构域与泛素相互作用。Pux10-1 T-DNA插入突变表型(脂滴在萌发过程中不会增大)可以由全长的AtPUX10补充，但不能通过缺失Uba结构域或Ubx结构域的截短版本来补充。在pux10-1突变体幼苗中，油球蛋白的降解通过Western blotting和串联质量标签的LC-MS/MS定量来减缓。通过使用GC-FID7测量标签水平，三酰甘油在pux10-1突变苗中的降解受到影响。AtPUX10对花粉管的生长很重要，因为它表明在体内花粉管的生长缓慢，突变的等位基因通过花粉的传递减少。总而言之，该提议的目的是更好地理解脂滴的分解，特别是在油球蛋白去除的背景下。

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.