Marx U, Polakowski T, Pomorski T, Lang C, Nelson H, Nelson N, Herrmann A
Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie/Biophysik, Germany.
Eur J Biochem. 1999 Jul;263(1):254-63. doi: 10.1046/j.1432-1327.1999.00497.x.
Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein.
有证据表明，内吞作用缺陷型的酿酒酵母end4酵母细胞能快速内化荧光磷脂类似物1-棕榈酰-2-{6-[7-硝基-2,1,3-苯并恶二唑-4-基（NBD）氨基]己酰}磷脂酰胆碱（P-C6-NBD-PtdCho）和P-C6-NBD-磷脂酰丝氨酸（P-C6-NBD-PtdSer）。在0℃时，这两种类似物在外质膜和细胞质膜小叶之间重新分布的半衰期均小于15分钟。内化类似物的平台期约为70%。跨双层运动可能是由蛋白质介导的，因为在由质膜脂质组成的脂质体中，这两种类似物的翻转都非常缓慢。细胞内ATP消耗约90%时，类似物的快速内化并未被消除。仅对于P-C6-NBD-PtdCho，观察到内化类似物的平台期适度下降约20%，而P-C6-NBD-PtdSer的平台期不受影响。Drs2蛋白在酿酒酵母end4细胞中类似物的快速跨双层运动中即使有作用也很小。在同时含有end4等位基因和drs2无效等位基因的酿酒酵母end4 Deltadrs2细胞中，在0℃下15分钟内，分别约有60%和50%的P-C6-NBD-PtdCho和P-C6-NBD-PtdSer被内化。通过膜联蛋白V的结合评估，P-C6-NBD-PtdSer向细胞质膜小叶的优先取向与内源性磷脂酰丝氨酸向细胞质膜小叶的隔离在性质上是一致的。实际上，未观察到膜联蛋白V与亲本野生型菌株或突变菌株的原生质体有结合。同样，通过用三硝基苯磺酸标记，未发现这些菌株之间内源性氨基磷脂向外质膜小叶的暴露有差异。因此，至少氨基磷脂的脂质不对称性在酿酒酵母end4细胞中得以保留，与Drs2蛋白的存在无关。
