Stevenson F T, Torrano F, Locksley R M, Lovett D H
Medical Service, San Francisco Veterans Administration Medical Center, California 94121.
J Cell Physiol. 1992 Aug;152(2):223-31. doi: 10.1002/jcp.1041520202.
Interleukin-1 (IL-1) is synthesized as a 31 kDa precursor protein, whose multiple extracellular activities are attributed to receptor binding of a processed, carboxy-terminal 17 kDa peptide. Unlike other secreted proteins, the IL-1 precursor lacks a hydrophobic leader sequence and is not found in organelles composing the classical secretory pathway. In order to further clarify the intracellular processing of IL-1, we studied its site of synthesis in human monocytes. Secreted and integral membrane proteins are translated on membrane-bound polyribosomes, while intracellular proteins are translated on free polyribosomes. Free and membrane-bound polysomes were isolated from Lipid A-stimulated monocyte lysates and immunoblotted using antibodies specific to the N-terminal regions of the IL-1 alpha and beta precursors. Free polysome fractions showed multiple small bands consistent with nascent peptide chains; membrane-bound polysomes yielded no detectable IL-1. Polysome fractions were then analyzed by immunoelectron microscopy; nascent IL-1 alpha and beta peptide chains were readily seen emerging from cytoskeletal-associated free polyribosomes, but not membrane-bound polyribosomes. Electron microscopic in situ hybridization revealed IL-1 mRNA chains attached to cytoskeletal-associated free, but not membrane-bound polyribosomes. The intracellular distribution of the fully synthesized IL-1 beta precursor was studied in human mesangial cells (HMC), whose cytoskeletal organization is more readily evaluated than that of monocytes. Dual immunofluorescence microscopy of these cells revealed a complex intracellular distribution of the fully synthesized 31 kDa IL-1 precursors. IL-1 was asymmetrically distributed between cytosolic, microtubule, and nuclear compartments, without association with actin or intermediate filaments. This demonstration of the sites of IL-1 synthesis and patterns of intracellular distribution provide further evidence for an extracellular release mechanism which is clearly distinct from the classical secretory pathway.
白细胞介素-1（IL-1）最初以31 kDa的前体蛋白形式合成，其多种细胞外活性归因于经过加工的羧基末端17 kDa肽与受体的结合。与其他分泌蛋白不同，IL-1前体缺乏疏水前导序列，并且在构成经典分泌途径的细胞器中未发现。为了进一步阐明IL-1的细胞内加工过程，我们研究了其在人单核细胞中的合成位点。分泌型和整合膜蛋白在膜结合多核糖体上翻译，而细胞内蛋白在游离多核糖体上翻译。从脂多糖刺激的单核细胞裂解物中分离出游离和膜结合的多核糖体，并用针对IL-1α和β前体N末端区域的抗体进行免疫印迹。游离多核糖体组分显示出多条与新生肽链一致的小条带；膜结合多核糖体未检测到IL-1。然后通过免疫电子显微镜分析多核糖体组分；很容易看到新生的IL-1α和β肽链从与细胞骨架相关的游离多核糖体中出现，但未从膜结合多核糖体中出现。电子显微镜原位杂交显示IL-1 mRNA链附着于与细胞骨架相关的游离多核糖体而非膜结合多核糖体。在人肾小球系膜细胞（HMC）中研究了完全合成的IL-1β前体的细胞内分布，其细胞骨架组织比单核细胞更容易评估。对这些细胞进行的双重免疫荧光显微镜检查显示，完全合成的31 kDa IL-1前体在细胞内呈复杂分布。IL-1不对称地分布在细胞质、微管和核区室之间，与肌动蛋白或中间丝无关。IL-1合成位点和细胞内分布模式的这一证明为一种明显不同于经典分泌途径的细胞外释放机制提供了进一步的证据。
