Muller W A, Steinman R M, Cohn Z A
J Cell Biol. 1980 Jul;86(1):304-14. doi: 10.1083/jcb.86.1.304.
Lactoperoxidase covalently coupled to latex spheres (LPO-latex) has been used to selectively iodinate the phagolysome (PL) membrane within living macrophages, as discussed in the accompanying article. This procedure labeled approximately 24 polypeptides in the PL membrane; these were similar to those iodinatable on the external surface of the plasma membrane (PM). We now report on the translocation and fate of these proteins when the cells are returned to culture. TCA-precipitable radioactivity was lost from cells with biphasic kinetics. 20-50% of the cell-associated radiolabel was rapidly digested (t 1/2 approximately equal to 1 h) and recovered in the culture medium as monoiodotyrosine. 50-80% of the label was lost slowly from cells ( 1/2 approximately equal to 24-30 h). Quantitative analysis of gel autoradiograms showed that all radiolabeled proteins were lost at the same rate in both the rapid and slow phases of digestion. Within 15-30 min aftr labeling of the PL membrane, EM autoradiography revealed that the majority of the cell-associated grains, which at time 0 were associated with PL, were now randomly dispersed over the plasmalemma. At this time, analysis of PM captured by a second phagocytic load revealed the presence of all labeled species originally present in the PL membrane. This demonstrated the rapid, synchronous centrifugal flow of PL polypeptides to the cell surface. Evidence was also obtained for the continuous influx of representative samples of the PM into the PL compartment by way of pinocytic vesicles. This was based on the constant flow of fluid phase markers into latex-containing PL and on the internalization of all iodinatable PM polypeptides into this locus. These observations provide evidence for the continuous, bidirectional flow of membrane polypeptides between the PM and the secondary lysosome and represent an example of a membrane flow and recycling mechanism.
如随附文章中所讨论的，与乳胶球共价偶联的乳过氧化物酶（LPO-乳胶）已被用于在活巨噬细胞内选择性地碘化吞噬溶酶体（PL）膜。该程序标记了PL膜中的约24种多肽；这些多肽与质膜（PM）外表面上可碘化的多肽相似。我们现在报告当细胞返回培养时这些蛋白质的转运和命运。三氯乙酸可沉淀的放射性以双相动力学从细胞中丢失。20%-50%与细胞相关的放射性标记物被快速消化（半衰期约为1小时），并以单碘酪氨酸的形式在培养基中回收。50%-80%的标记物从细胞中缓慢丢失（半衰期约为24-30小时）。凝胶放射自显影片的定量分析表明，所有放射性标记的蛋白质在消化的快速和缓慢阶段都以相同的速率丢失。在PL膜标记后15-30分钟内，电子显微镜放射自显影显示，大多数与细胞相关的颗粒，在时间为0时与PL相关，现在随机分散在质膜上。此时，对第二次吞噬负载捕获的PM的分析揭示了PL膜中最初存在 的所有标记物种的存在。这证明了PL多肽向细胞表面的快速、同步离心流动。还获得了证据，表明PM的代表性样品通过胞饮小泡持续流入PL区室。这是基于液相标记物持续流入含乳胶的PL以及所有可碘化的PM多肽内化到该位点。这些观察结果为PM和次级溶酶体之间膜多肽的持续双向流动提供了证据，并代表了膜流动和循环机制的一个例子。
