Roles of endosomal lipid sorting in membrane degradation

生物膜的脂质组成对其功能特性至关重要。它在溶酶体内的膜降解过程中也起重要作用。众所周知，除了适当的脂质组合物，不同的脂质转移蛋白的存在下，本地化的内体/溶酶体室，需要膜崩解和degradation.Within的最后一个资助期内，我们开发了一种在体外测定系统的蛋白质介导的脂质双层之间的转移和自发的，以及蛋白质介导的膜融合。我们发现，在存在双（单酰基甘油）磷酸盐（BMP），内体和溶酶体内膜的酸性脂质特性，神经酰胺刺激胆固醇转移介导的尼曼-匹克C2蛋白（NPC 2），而鞘磷脂发挥了强烈的抑制作用。基于这些体外数据，我们开发了一种脂质分选和膜降解模型，根据该模型（除其他特征外），内体膜的组成通过快速作用酶酸性鞘磷脂酶调节降解，酸性鞘磷脂酶将抑制性脂质鞘磷脂转化为刺激性脂质神经酰胺，进而以非常受控的方式在溶酶体中更下游降解。为了证明这种变化的脂质组成的内吞途径发生在细胞和组织中，我们计划subfractionation实验分离质膜馏分，早期内体，晚期内体，和溶酶体，并确定膜组成中发现这些细胞器的薄层色谱和质谱。我们还将进行标记实验，然后进行显微镜技术，以确认脂质分析的结果。我们假设，最适合膜降解的脂质组合物建立在这一途径中。稍后，我们将使用具有实验确定的脂质组成的膜进行转移和融合测定，以生成溶酶体膜降解过程的详细分子模型。

The lipid composition of biological membranes is crucial for their functional properties. It also plays an important role during membrane degradation within the lysosomes. It is known that in addition to an appropriate lipid composition, the presence of different lipid transfer proteins, localized in the endosomal / lysosomal compartment, is required for membrane disintegration and degradation.Within the last funding period, we developed an in vitro assay-system for protein-mediated lipid transfer between bilayers and for spontaneous as well as for protein-mediated membrane fusion. We showed that in the presence of bis(monoacylglycero)phosphate (BMP), an acidic lipid characteristic for intraendosomal and intralysosomal membranes, ceramide stimulates cholesterol-transfer mediated by the Niemann-Pick C2-protein (NPC2), while sphingomyelin exerts a drastic inhibitory effect. Based on these in vitro data we developed a model for lipid sorting and membrane degradation, according to that (among other features), the composition of intraendosomal membranes is adjusted for degradation by the rapidly acting enzyme, acid sphingomyelinase, which converts the inhibiting lipid sphingomyelin to the stimulating lipid ceramide, which, in turn, is degraded more downstream in lysosomes in a very controlled way. To demonstrate that this change in lipid composition during the endocytic pathway occurrs in cells and tissues, we plan subfractionation experiments to isolate plasma membrane fractions, early endosomes, late endosomes, and lysosomes and to determine the membrane composition found in these organelles by thin layer chromatography and mass spectrometry. We will also perform labeling experiments followed by microscopical techniques to confirm the results from lipid analysis. We assume that lipid compositions optimal for membrane degradation are established in this pathway. Later on, we will conduct transfer and fusion assays with membranes having the experimentally determined lipid composition to generate a detailed molecular model for the process of lysosomal membrane degradation.