Vey M, Pilkuhn S, Wille H, Nixon R, DeArmond S J, Smart E J, Anderson R G, Taraboulos A, Prusiner S B
Department of Neurology, University of California, San Francisco 94143, USA.
Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14945-9. doi: 10.1073/pnas.93.25.14945.
Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrPSc) interacts with the substrate cellular PrP (PrPC) during conversion into nascent PrPSc. While PrPSc appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrPC is converted into PrPSc. We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cells contain PrPC and PrPSc. After lysis of ScN2a cells in ice-cold Triton X-100, both PrP isoforms and an N-terminally truncated form of PrPC (PrPC-II) were found concentrated in detergent-insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H-ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N-hydroxysuccinimide-biotin, both PrPC and PrPSc were found biotinylated in CLD fractions. Similar results on the colocalization of PrPC and PrPSc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrPC and PrPSc are present in CLDs and, thus, support the hypothesis that the PrPSc formation occurs within this subcellular compartment.
转基因研究结果表明，朊病毒蛋白（PrPSc）的瘙痒病异构体在转化为新生PrPSc的过程中与底物细胞PrP（PrPC）相互作用。虽然PrPSc似乎主要在溶酶体中积累，但有人提出类似小窝的结构域（CLDs）是PrPC转化为PrPSc的场所。我们在此报告，从感染瘙痒病的神经母细胞瘤（ScN2a）细胞中分离出的CLDs含有PrPC和PrPSc。在冰冷的Triton X-100中裂解ScN2a细胞后，发现两种PrP异构体和一种N端截短形式的PrPC（PrPC-II）都集中在类似于CLDs的去污剂不溶性复合物中，这些复合物通过在蔗糖梯度中浮选分离得到。当通过超声处理和梯度离心从质膜中纯化CLDs时，也得到了类似的结果；采用此方法不使用去污剂，从而将可能因蛋白质在亚细胞组分之间重新分布而产生的假象降至最低。发现小窝标记物神经节苷脂GM1和H-ras集中在CLD组分中。当用非渗透性试剂磺基-N-羟基琥珀酰亚胺-生物素标记质膜蛋白时，发现CLD组分中的PrPC和PrPSc都被生物素化。从叙利亚仓鼠脑中分离CLDs时，在PrPC和PrPSc共定位方面也得到了类似的结果。我们的研究结果表明，PrPC和PrPSc都存在于CLDs中，因此支持了PrPSc形成发生在这个亚细胞区室中的假说。
