Menon A K, Hrafnsdóttir S
Department of Biochemistry, University of Wisconsin-Madison, Madison, 53706-1569, USA.
Curr Biol. 2000 Mar 9;10(5):241-52. doi: 10.1016/s0960-9822(00)00356-0.
A long-standing problem in understanding the mechanism by which the phospholipid bilayer of biological membranes is assembled concerns how phospholipids flip back and forth between the two leaflets of the bilayer. This question is important because phospholipid biosynthetic enzymes typically face the cytosol and deposit newly synthesized phospholipids in the cytosolic leaflet of biogenic membranes such as the endoplasmic reticulum (ER). These lipids must be transported across the bilayer to populate the exoplasmic leaflet for membrane growth. Transport does not occur spontaneously and it is presumed that specific membrane proteins, flippases, are responsible for phospholipid flip-flop. No biogenic membrane flippases have been identified and there is controversy as to whether proteins are involved at all, whether any membrane protein is sufficient, or whether non-bilayer arrangements of lipids support flip-flop.
To test the hypothesis that specific proteins facilitate phospholipid flip-flop in the ER, we reconstituted transport-active proteoliposomes from detergent-solubilized ER vesicles under conditions in which protein-free liposomes containing ER lipids were inactive. Transport was measured using a synthetic, water-soluble phosphatidylcholine and was found to be sensitive to proteolysis and associated with proteins or protein-containing complexes that sedimented operationally at 3.8S. Chromatographic analyses indicated the feasibility of identifying the transporter(s) by protein purification approaches, and raised the possibility that at least two different proteins are able to facilitate transport. Calculations based on a simple reconstitution scenario suggested that the transporters represent approximately 0.2% of ER membrane proteins.
Our results clearly show that specific proteins are required to translocate a phosphatidylcholine analogue across the ER membrane. These proteins are likely to be the flippases, which are required to translocate natural phosphatidylcholine and other phospholipids across the ER membrane. The methodology that we describe paves the way for identification of a flippase.
在理解生物膜磷脂双分子层组装机制方面，一个长期存在的问题是磷脂如何在双分子层的两个小叶之间来回翻转。这个问题很重要，因为磷脂生物合成酶通常面向胞质溶胶，并将新合成的磷脂沉积在生物膜（如内质网，ER）的胞质小叶中。这些脂质必须穿过双分子层以填充外质小叶，从而实现膜的生长。这种转运不会自发发生，推测特定的膜蛋白——翻转酶——负责磷脂的翻转。尚未鉴定出生物膜翻转酶，对于是否真的有蛋白质参与、任何一种膜蛋白是否足够，或者脂质的非双分子层排列是否支持翻转，都存在争议。
为了验证内质网中特定蛋白质促进磷脂翻转这一假设，我们在不含蛋白质的内质网脂质脂质体无活性的条件下，从去污剂溶解的内质网囊泡中重构了具有转运活性的蛋白脂质体。使用一种合成的水溶性磷脂酰胆碱来测量转运，发现其对蛋白水解敏感，并且与在操作上沉降系数为3.8S的蛋白质或含蛋白质复合物相关。色谱分析表明通过蛋白质纯化方法鉴定转运蛋白是可行的，并提出至少有两种不同蛋白质能够促进转运的可能性。基于一个简单重构方案的计算表明，转运蛋白约占内质网膜蛋白的0.2%。
我们的结果清楚地表明，需要特定蛋白质才能使磷脂酰胆碱类似物穿过内质网膜。这些蛋白质可能就是翻转酶，它们是使天然磷脂酰胆碱和其他磷脂穿过内质网膜所必需的。我们所描述的方法为鉴定翻转酶铺平了道路。
