Pugsley A P, Kornacker M G, Ryter A
Unité de Génétique Moléculaire, Institut Pasteur, Paris, France.
Mol Microbiol. 1990 Jan;4(1):59-72. doi: 10.1111/j.1365-2958.1990.tb02015.x.
Three different techniques, protease accessibility, cell fractionation and in situ immunocytochemistry, were used to study the location of the lipoprotein pullulanase produced by Escherichia coli K12 carrying the cloned pullulanase structural gene (pulA) from Klebsiella pneumoniae, with or without the K. pneumoniae genes required to transport pullulanase to the cell surface (secretion-competent and secretion-incompetent, respectively). Pullulanase produced by secretion-competent strains could be slowly but quantitatively released into the medium by growing the cells in medium containing pronase. The released pullulanase lacked the N-terminal fatty-acylated cysteine residue (and probably also a short N-terminal segment of the pullulanase polypeptide), confirming that the N-terminus is the sole membrane anchor in the protein. Pullulanase produced by secretion-incompetent strains was not affected by proteases, confirming that it is not exposed on the cell surface. Pullulanase cofractionated with both outer and inner membrane vesicles upon isopycnic sucrose gradient centrifugation, irrespective of the secretion competence of the strain. Examination by electronmicroscopy of vesicles labelled with antipullulanase serum and protein A-gold confirmed that pullulanase was associated with both types of vesicles. When thin-sectioned cells were examined by the same technique, pullulanase was found to be located mainly on the cell surface of the secretion-competent cells and mainly in the proximity of the inner membrane in the secretion-incompetent cells. Thus, while the results from three independent techniques (substrate accessibility, protease accessibility and in situ immunocytochemistry) show that pullulanase is transported to the cell surface of secretion-competent cells, this could not be confirmed by cell-fractionation techniques. Possible explanations for this discrepancy are discussed.
采用三种不同技术，即蛋白酶可及性分析、细胞分级分离和原位免疫细胞化学，研究了携带来自肺炎克雷伯菌的克隆支链淀粉酶结构基因（pulA）的大肠杆菌K12产生的支链淀粉酶的定位情况，该大肠杆菌分别带有或不带有将支链淀粉酶转运至细胞表面所需的肺炎克雷伯菌基因（分别为分泌能力正常和分泌能力缺陷型）。通过在含有链霉蛋白酶的培养基中培养细胞，分泌能力正常的菌株产生的支链淀粉酶可缓慢但定量地释放到培养基中。释放的支链淀粉酶缺乏N端脂肪酰化半胱氨酸残基（可能还缺少支链淀粉酶多肽的一段短N端片段），这证实了N端是该蛋白唯一的膜锚定部位。分泌能力缺陷型菌株产生的支链淀粉酶不受蛋白酶影响，这证实它未暴露在细胞表面。在等密度蔗糖梯度离心中，无论菌株的分泌能力如何，支链淀粉酶都与外膜和内膜囊泡共分级分离。用抗支链淀粉酶血清和蛋白A-金标记囊泡，通过电子显微镜检查证实支链淀粉酶与这两种类型的囊泡都有关联。当用相同技术检查薄切片细胞时，发现支链淀粉酶主要位于分泌能力正常的细胞的细胞表面，而在分泌能力缺陷型细胞中主要位于内膜附近。因此，虽然三种独立技术（底物可及性、蛋白酶可及性和原位免疫细胞化学）的结果表明支链淀粉酶被转运至分泌能力正常的细胞的细胞表面，但细胞分级分离技术无法证实这一点。本文讨论了这种差异的可能解释。
