Seglen P O, Gordon P B
J Cell Biol. 1984 Aug;99(2):435-44. doi: 10.1083/jcb.99.2.435.
Sequestration of the inert cytosolic marker [14C]sucrose by sedimentable organelles was measured in isolated rat hepatocytes made transiently permeable to sucrose by means of electropermeabilization. Lysosomal integrity, protein degradation, autophagic sequestration, and other cellular functions were not significantly impaired by the electric treatment. Hepatocytes sequestered sucrose at an initial rate of approximately 10%/h, which is threefold higher than the estimated rate of autophagic-lysosomal protein degradation. Almost one-third would appear to represent mitochondrial fluid uptake; the rest was nearly completely and specifically inhibited by 3-methyladenine (3MA) and can be regarded as autophagic sequestration. A complete amino acid mixture was somewhat less inhibitory than 3MA, and partially antagonized the effect of the latter. This paradoxical effect, taken together with the high sequestration rate, may suggest heterogeneity as well as selectivity in autophagic sequestration. There was no detectable recycling of sequestered [14C]sucrose between organelles and cytosol. Studies of individual amino acids revealed histidine as the most effective sequestration inhibitor. Leucine may have a regulatory function, as indicated by its unique additive/synergistic effect, and a combination of Leu + His was as effective as the complete amino acid mixture. Asparagine inhibited sequestration only 20%, i.e., its very strong effect on overall (long-lived) protein degradation must partially be due to post-sequestrational inhibition. The lysosomal (amine-sensitive) degradation of short-lived protein was incompletely inhibited by 3MA, indicating a contribution from nonautophagic processes like crinophagy and endocytic membrane influx. The ability of an amino acid mixture to specifically antagonize the inhibition of short-lived protein degradation by AsN + GIN (but not by 3MA) may suggest complex amino acid interactions at the level of fusion between lysosomes and other vesicles in addition to the equally complex interactions at the level of autophagic sequestration.
通过电穿孔使分离的大鼠肝细胞对蔗糖短暂通透，以此来测定可沉降细胞器对惰性胞质标记物[14C]蔗糖的隔离情况。电处理对溶酶体完整性、蛋白质降解、自噬隔离及其他细胞功能并无显著损害。肝细胞隔离蔗糖的初始速率约为10%/小时，这比自噬溶酶体蛋白质降解的估计速率高三倍。几乎三分之一似乎代表线粒体液体摄取；其余部分几乎完全且特异性地被3 - 甲基腺嘌呤（3MA）抑制，可被视为自噬隔离。完整氨基酸混合物的抑制作用比3MA稍弱，并部分拮抗了后者的作用。这种矛盾的效应，连同高隔离率，可能表明自噬隔离存在异质性和选择性。在细胞器和胞质溶胶之间未检测到隔离的[14C]蔗糖的再循环。对单个氨基酸的研究表明，组氨酸是最有效的隔离抑制剂。亮氨酸可能具有调节功能，如其独特的相加/协同效应所示，亮氨酸 + 组氨酸的组合与完整氨基酸混合物的效果相同。天冬酰胺仅抑制隔离20%，即其对整体（长寿）蛋白质降解的极强作用一定部分归因于隔离后抑制。3MA对短命蛋白质的溶酶体（胺敏感）降解抑制不完全，表明诸如分泌自噬和内吞膜内流等非自噬过程也有作用。氨基酸混合物特异性拮抗天冬酰胺 + 谷氨酰胺（而非3MA）对短命蛋白质降解抑制的能力，可能表明除了自噬隔离水平上同样复杂的相互作用外，在溶酶体与其他囊泡融合水平上也存在复杂的氨基酸相互作用。
