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

含UBX结构域的支架蛋白FAF1/PUX10在幼苗和花粉管脂滴降解中的功能

基本信息

批准号：
290136108
负责人：
Professor Dr. Till Ischebeck
金额：
--
依托单位：
Arbeitsgruppe Plant Biochemistry and Biotechnology
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/290136108?language=en
关键词：
function UBX domain containing scaffold

项目摘要

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.

脂滴是植物中重要的贮藏细胞器，尤其在花粉和种子中。与这些细胞器相关的主要蛋白质是油蛋白。油蛋白阻止脂滴融合，它们的丰度决定了脂滴的大小。在种子萌发过程中，油苷被泛素化，随后被降解。由于这种降解比油本身的降解快，因此脂滴的大小增加。我们在烟草花粉管脂滴上鉴定了一个支架蛋白NtFAF1/NtPUX10。其拟南芥同源物AtPUX10在烟草花粉管中短暂表达时，也能靶向脂滴。从哺乳动物酵母中的同源物中，我们知道这些蛋白结合泛素化蛋白并招募aaa型atp酶（酵母中的Cdc48p），该酶可以展开蛋白质并将其从膜上移除。由于我们还可以证明植物PUX10蛋白可以将拟南芥AtCDC48a招募到脂滴上，我们怀疑这整个复合物可能参与了从脂滴中去除泛素化油苷的过程。在两个独立的pux10拟南芥突变系中，我们可以发现，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蛋白没有被去除，从而阻止了脂滴的融合。为了进一步支持我们的模型，我们想证明：1。在拟南芥中，AtPUX10在内在启动子下表达，定位到种子脂滴上。AtPUX10通过其UBX结构域直接与所有三个AtCDC48亚型相互作用，使用全长AtPUX10和烟草花粉管中截断的AtPUX10进行瞬时bbc测定。使用全长AtPUX10和截断版本的y2h检测，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（在萌发过程中脂滴大小不增加）可以由全长AtPUX10补充，但不能由缺失uba -结构域或ubx -结构域的截断版本补充。通过western blotting和串联质谱标记（LC-MS/ ms）的定量分析，pux10-1突变体幼苗的油苷降解速度减慢。通过GC-FID7测定tag水平，研究pux10-1突变体幼苗中甘油三酯降解的影响。AtPUX10对花粉管生长很重要，表明体内花粉管生长减慢，突变等位基因通过花粉的传播减少。总而言之，该建议旨在更好地理解脂滴分解，特别是在去除油蛋白的背景下。