Characterization of triacylglycerol biosynthesis in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora

Manoj G Pillai , Milan Certik , Toro Nakahara, Yasushi Kamisaka
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引用次数: 30

Abstract

Triacylglycerol (TG) biosynthetic enzymes were characterized in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora. When the membrane or lipid body fraction of this fungus was incubated with [14C]oleoyl-CoA without adding exogenous acyl acceptors, radioactivity was incorporated predominantly into TG, indicating that diacylglycerol acyltransferase (DGAT) used endogenous diacylglycerol to incorporate [14C]oleoyl-CoA into TG. Adding glycerol 3-phosphate or lysophosphatidic acid increased radiolabeled phosphatidic acid (PA) in the membrane fraction, which reflected the presence of glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT). Label accumulation did not occur in lysophosphatidic acid when glycerol 3-phosphate was added, suggesting that GPAT was rate-limiting in sequential acylation. In the lipid body fraction, adding lysophosphatidic acid similarly increased radiolabeled PA, whereas adding glycerol 3-phosphate caused much lower increase in radiolabeled PA. Quantitative assays for GPAT, LPAAT, phosphatidic acid phosphatase (PAP), and DGAT essentially confirmed the results obtained from [1-14C]oleoyl-CoA incorporation; LPAAT had the highest activity in the membrane and lipid body fractions, GPAT was significantly lower in the lipid body fraction, and DGAT was much higher in the lipid body fraction. GPAT and LPAAT in the membrane fraction had a strong preference toward oleoyl-CoA as a substrate over palmitoyl-CoA. Results indicate that TG biosynthetic enzymes had different subcellular distribution with the sequence of enrichment in the lipid body fraction, i.e., GPAT<LPAAT≈PAP<DGAT. This may reflect a TG biosynthetic process from endoplasmic reticulum membranes to lipid bodies in the fungus.

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产油真菌拉曼尼亚Mortierella var. angulispora亚细胞组分中三酰基甘油生物合成的表征
研究了产油真菌拉曼尼亚Mortierella ramanniana var. angulispora亚细胞组分中甘油三酯(Triacylglycerol, TG)生物合成酶的特征。当该真菌的膜或脂质体部分与[14C]油基辅酶a孵育而不添加外源性酰基受体时,放射性主要被掺入TG,表明二酰基甘油酰基转移酶(DGAT)利用内源性二酰基甘油将[14C]油基辅酶a掺入TG。添加3-磷酸甘油或溶血磷脂酸增加了膜组分中放射性标记磷脂酸(PA),反映了甘油-3-磷酸酰基转移酶(GPAT)和溶血磷脂酸酰基转移酶(LPAAT)的存在。当加入3-磷酸甘油时,溶血磷脂酸中没有发生标记积累,表明GPAT在顺序酰化中具有限速作用。在脂质体部分,添加溶血磷脂酸同样增加了放射性标记PA,而添加甘油3-磷酸对放射性标记PA的增加作用要小得多。GPAT、LPAAT、磷脂酸磷酸酶(PAP)和DGAT的定量分析基本上证实了[1-14C]油基coa掺入的结果;LPAAT在膜和脂质体组分中活性最高,GPAT在脂质体组分中活性显著降低,而DGAT在脂质体组分中活性显著升高。相对于棕榈酰辅酶a,膜组分中的GPAT和LPAAT更倾向于油酰辅酶a作为底物。结果表明,TG生物合成酶在脂质体部分的富集顺序不同,其亚细胞分布为GPAT<LPAAT≈PAP<DGAT。这可能反映了真菌从内质网膜到脂质体的TG生物合成过程。
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