Diaz R, Wileman T E, Anderson S J, Stahl P
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110.
Biochem J. 1989 May 15;260(1):127-34. doi: 10.1042/bj2600127.
The mannose receptor mediates the transport of high-mannose glycoproteins from the cell surface to lysosomes in macrophages. The binding of ligand to the receptor is dependent on both pH and Ca2+. Upon internalization, ligands enter an acidic pre-lysosomal compartment where receptor-ligand dissociation takes place. Acidification is driven by an endosomal proton pump and anion transport is coupled to this acidification step. A permeabilized-cell assay has been designed to characterize the ionic requirements for receptor-ligand dissociation in endosomes. The plasma membrane of macrophages has been permeabilized selectively with digitonin without affecting endosomal membranes. Receptor-ligand dissociation in permeabilized cells required ATP and was blocked by proton ionophores. Di-isothiocyanostilbene-disulphonic acid and N-ethylmaleimide also blocked dissociation, but mitochondrial ATPase inhibitors and vanadate were ineffective. To explore the nature of the anion requirement for acidification, the ability of different anions to compensate for Cl- was tested. For the halide series, Br- was as equally effective as Cl- in supporting receptor-ligand dissociation, but I- was inhibitory. Citrate and gluconate were only partially effective, while SO4(2-), NO3- and PO4(2-) blocked dissociation. Addition of Ca2+ to permeabilized-cell preparations impaired ATP-dependent dissociation without affecting endosome acidification. These results suggest that the endosomal membrane has a Ca2+ conductance that would permit the rapid efflux of Ca2+ from endosomes during acidification, and this would appear to be a necessary step for efficient sorting of Ca2+-dependent receptors from their ligands.
甘露糖受体介导巨噬细胞中高甘露糖糖蛋白从细胞表面向溶酶体的转运。配体与受体的结合既依赖于pH值，也依赖于Ca2+。内化后，配体进入酸性的前溶酶体区室，在那里发生受体 - 配体解离。酸化由内体质子泵驱动，阴离子转运与这一酸化步骤偶联。已设计一种通透细胞测定法来表征内体中受体 - 配体解离的离子需求。巨噬细胞的质膜已用洋地黄皂苷选择性通透，而不影响内体膜。通透细胞中的受体 - 配体解离需要ATP，并被质子离子载体阻断。二异硫氰酸芪二磺酸和N - 乙基马来酰亚胺也阻断解离，但线粒体ATP酶抑制剂和钒酸盐无效。为了探究酸化对阴离子需求的本质，测试了不同阴离子补偿Cl-的能力。对于卤化物系列，Br-在支持受体 - 配体解离方面与Cl-同样有效，但I-具有抑制作用。柠檬酸盐和葡萄糖酸盐仅部分有效，而SO4(2-)、NO3-和PO4(2-)阻断解离。向通透细胞制剂中添加Ca2+会损害ATP依赖性解离，而不影响内体酸化。这些结果表明，内体膜具有Ca2+电导率，这将允许在酸化过程中Ca2+从内体快速流出，这似乎是从其配体中有效分选Ca2+依赖性受体的必要步骤。
