Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts.
Sorting of an internalized plasma membrane lipid between recycling and degradative pathways in normal and Niemann-Pick, type A fibroblasts.
复制标题
DOI：
10.1083/jcb.111.2.429
复制
发表时间：
1990-08
影响因子：
7.8
通讯作者：
Pagano, R E
中科院分区：
文献类型：
--
作者：
Koval, M;Pagano, R E
我们研究了神经鞘磷脂类似物N-(N-[6-[(7-硝基-2-氧-1，3-二唑-4-基)氨基]己基])-鞘氨醇磷酸胆碱(C6-NBD-SM)在正常人和Niemann-Pick A型(NP-A)人皮肤成纤维细胞单层中的代谢和细胞内转运。C6-NBD-SM通过将C6-NBD-SM单体从脂质体转移到细胞中，在7℃下整合到质膜双层中。洗涤细胞，在37℃升温3min内，正常和NP-A成纤维细胞都内化了质膜上的C6-NBD-SM，导致细胞内荧光点状。在正常和NP-A细胞中，C6-NBD-SM内化和从细胞内室转运到质膜(循环)的速率相似。随着37℃下时间的延长，内化的C6-NBD-SM积聚在NP-A成纤维细胞的溶酶体中，而正常成纤维细胞的高尔基体荧光增强，未见溶酶体标记。由于NP-A成纤维细胞缺乏溶酶体(酸性)鞘磷脂酶(A-sMase)，这一结果表明C6-NBD-SM的水解阻止了其沿降解途径在正常成纤维细胞的溶酶体中积聚。我们用A-sMase在正常细胞中对C6-NBD-SM的水解量作为C6-NBD-SM从细胞表面转运到溶酶体的量度。在一段滞后期后，C6-NBD-SM以大约8%/h的速度递送到溶酶体，这一速度大约是C6-NBD-SM从细胞内室循环到质膜的速度的18-19倍。因此，少量的C6-NBD-SM沿着降解途径运输，而大多数内吞的C6-NBD-SM被分拣沿着质膜循环途径运输。
We examined the metabolism and intracellular transport of a fluorescent sphingomyelin analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4- yl)amino]caproyl])- sphingosylphosphorylcholine (C6-NBD-SM), in both normal and Niemann-Pick, type A (NP-A) human skin fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 3 min of warming to 37 degrees C, both normal and NP-A fibroblasts had internalized C6-NBD-SM from the plasma membrane, resulting in a punctate pattern of intracellular fluorescence. Rates for C6-NBD-SM internalization and transport from intracellular compartments to the plasma membrane (recycling) were similar for normal and NP-A cells. With increasing time at 37 degrees C, internalized C6-NBD-SM accumulated in the lysosomes of NP-A fibroblasts, while normal fibroblasts showed increasing Golgi apparatus fluorescence with no observable lysosomal labeling. Since NP-A fibroblasts lack lysosomal (acid) sphingomyelinase (A-SMase), this result suggested that hydrolysis of C6-NBD-SM prevented its accumulation in the lysosomes of normal fibroblasts during its transport along the degradative pathway. We used the amount of C6-NBD- SM hydrolysis by A-SMase in normal cells as a measure of C6-NBD-SM transported from the cell surface to the lysosomes. After a lag period, C6-NBD-SM was delivered to the lysosomes at a rate of approximately 8%/h. This rate was approximately 18-19 fold slower than the rate of C6- NBD-SM recycling from intracellular compartments to the plasma membrane. Thus, small amounts of C6-NBD-SM were transported along the degradative pathway, while most endocytosed C6-NBD-SM was sorted for transport along the plasma membrane recycling pathway.