Roles of FREE1 in regulating lipid droplet turnover in arabidopsis

Lipid droplets (LDs) stored during seed development are mobilized and provide essential lipid derived molecules to support seedling growth upon germination. Triacylglycerol (TAG) is the main component of neutral lipids stored in LDs. The lipase Sugar Dependent 1 (SDP1) in Arabidopsis thaliana hydrolyzes TAGs, is localized on peroxisomes and traffics to LDs through retromer-regulated peroxisome extension. The molecular machinery underlying the targeting of SDP1 to LDs remains elusive. R ecently, it has demonstrated that the plant unique protein termed FREE1 ( FYVE domain protein required for endosomal sorting 1) play important roles in regulating multivesicular body (MVB) biogenesis and MVB mediated vacuolar trafficking in plants. The loss of functional FREE1 caused the defective MVB formation, fragmented vacuole, and accumulated autophagasomes. The Arabidopsis free1 mutant has seedling lethality. Using a combination of cellular, molecular and genetic approaches, I demonstrate a novel function of a plant-unique ESCRT (endosomal sorting complex required for transport) component FREE1 in regulating peroxisome tubulation-mediated LDs turnover in plants. I have demonstrated that during early stages of germination LD degradation is impaired in free1 mutant, and degradation of protein LDAP3 is also impaired in free1 mutant, whereas the tubulation of SDP1 marked peroxisomes and the proper trafficking of the SDP1 to the surface of LDs are also altered in the free1 mutant, moreover the tubulation of PEX11E marked peroxisomes are also affected in the free1 mutant. Live cell imaging and biochemical analysis further showed that FREE1 is dynamically associated with SDP1 and interacts with SDP1 to regulate SDP1 targeting to LDs surface to perform its activity. Whole cell electron tomography analysis highlights the membrane dynamics and contacts between peroxisome tubules and the engulfed LDs in WT vs. free1 mutant. Taken together, this study demonstrates a pivotal role of FREE1 in mediating LDs degradation in germinating seedlings through regulating peroxisome tubulation and SDP1 targeting, thus provide the first example of ESCRT functions in a non-endosomal organelle, the peroxisome, in eukaryotes.種子發育過程中存儲的脂滴（LDs ）被水解代謝提供能量以及必需的脂質衍生分子以支持發芽後的幼苗生長。三酰基甘油（ TAG ）是 LD 中存儲的主要中性脂質成分。擬南芥中的糖依賴性脂肪酶 1 SDP1 ）其主要功能是催化水解 TAG 。 SDP1 定位於過氧化物酶體上，並通過後體 (retromer) 調控的過氧化物酶體延伸轉運至脂滴的表面。 SDP1 靶向脂滴表面的分子機制仍然未證明清楚。 最近發現了一個植物特異的 ESCRT （轉運所需的內體分選複合物）組分稱為 FREE1 （內體分選所需的 FYVE結構域蛋白 1 ），並證明 FREE1 在調節植物膜運輸和多泡體（ MVB ）生成過程起多重作用。 FREE1 蛋白缺失會導致多泡體（ MVB ）生成受損，液泡合成受損，自噬體積累。 在這裡， 結合細胞，分子和遺傳方法，我們證明了植物特異的 ESCRT （運輸所需的內體分揀複合體）組件稱為 FREE1 在調節植物中過氧化物酶體微管介導的脂滴代謝中的新功能。我們已經證明，在發芽的早期階段， free1 突變體的脂滴降解受到阻止， LDAP3 蛋白降解在 free1 突變體中受損， 並且 free1 突變體中 SDP1 標記的過氧化物酶體 的微管形成和 SDP1 轉運到脂滴表面的過程也發生了改變 以及PEX11 標記的過氧化物酶體的微管形成 。激光共聚焦活細胞成像和生化分析進一步表明， FREE1 與 SDP1 動態相關並與 SDP1 相互作用，以調節 SDP1 靶向到脂滴表面以執行 SDP1 的脂酶活性。全細胞電子斷層掃描分析突出了 WT 與 free1 突變體中過氧化物酶體小管與被吞噬的脂滴之間的動態過程。綜上所述，我們的研究表明，FREE1 通過調節過氧化物酶體管和 SDP1 靶向介導發芽幼苗中脂滴的降解發揮了關鍵作用，從而在真核生物的非內體細胞器過氧化物酶體中提供了 ESCRT 功能的第一個實例。Huang, Shuxian.Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves ).Abstracts also in Chinese.Title from PDF title page (viewed on …)