Klein D P, Galea S, Jongstra J
Department of Immunology, University of Toronto, Ontario, Canada.
J Immunol. 1990 Nov 1;145(9):2967-73.
LSP1 is a lymphocyte-specific intracellular Ca2(+)-binding protein. We found previously that a fraction of the total cellular pool of LSP1 protein accumulates at or near the cytoplasmic face of the plasma membrane. LSP1 protein was also shown to be present in the cytoplasm. Here we report that approximately 10% of the total intracellular LSP1 protein is associated with the Nonidet P-40 insoluble cytoskeleton of the mIgM+, mIgD+ B lymphoma cell line BAL17. Variation in conditions of extraction did not alter this value. To rule out the possibility that LSP1 associates with the nucleus that is also present in the detergent insoluble pellet, we prepared a separate nuclear fraction essentially free of cytoskeletal material and found only trace amounts of LSP1 protein. After accounting for yield losses during subcellular fractionation by measuring the recovery of 125I-labeled membrane IgM, or of the cytoplasmic marker enzyme lactate dehydrogenase activity, the LSP1 in membrane fractions was calculated to represent approximately 30% of the total cellular LSP1 and cytoplasmic LSP1 accounted for approximately 55% of the total. Approximately 75% of the plasma membrane LSP1 protein was soluble in 1% Nonidet P-40 containing buffer, indicating that the majority of the LSP1 in the plasma membrane fraction was distinct from the cytoskeletal LSP1 protein. The preparation of membrane fractions in the presence of 1 M NaCl, or washing of membranes in 3 M KCl did not diminish the levels of membrane LSP1. These results show the existence of three discrete intracellular LSP1 pools. Double label immunofluorescence studies showed that the peripheral ring-like distribution of LSP1 in BAL17 cells became a distinct cap upon cross-linking the mIgM. These intracellular LSP1 caps were always found to be located directly underneath the mIgM caps.
LSP1是一种淋巴细胞特异性的细胞内钙离子结合蛋白。我们之前发现，LSP1蛋白的一部分会在细胞膜的胞质面或其附近积累。LSP1蛋白也存在于细胞质中。在此我们报告，在mIgM+、mIgD+ B淋巴瘤细胞系BAL17中，约10%的细胞内总LSP1蛋白与非离子去污剂P-40不溶性细胞骨架相关。提取条件的变化并未改变这一数值。为排除LSP1与去污剂不溶性沉淀中也存在的细胞核相关的可能性，我们制备了一个基本不含细胞骨架物质的单独核部分，仅发现痕量的LSP1蛋白。通过测量125I标记的膜IgM或细胞质标记酶乳酸脱氢酶活性的回收率来计算亚细胞分级分离过程中的产量损失后，膜部分中的LSP1经计算约占细胞总LSP1的30%，细胞质LSP1约占总量的55%。约75%的质膜LSP1蛋白可溶于含1%非离子去污剂P-40的缓冲液中，这表明质膜部分中的大多数LSP1与细胞骨架LSP1蛋白不同。在1 M NaCl存在下制备膜部分，或用3 M KCl洗涤膜，均未降低膜LSP1的水平。这些结果表明存在三个离散的细胞内LSP1池。双标记免疫荧光研究表明，BAL17细胞中LSP1的外周环状分布在mIgM交联后变成一个明显的帽状结构。这些细胞内LSP1帽总是直接位于mIgM帽的下方。
