Cardelli J A, Richardson J, Miears D
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130.
J Biol Chem. 1989 Feb 25;264(6):3454-63.
Radiolabel pulse-chase and subcellular fractionation procedures were used to analyze the transport, proteolytic processing, and sorting of two lysosomal enzymes in Dictyostelium discoideum cells treated with the weak bases ammonium chloride and chloroquine. Dictyostelium lacks detectable cation-independent mannose-6-phosphate receptors and represents an excellent system to investigate alternative mechanisms for lysosomal enzyme targeting. Exposure of growing cells to ammonium chloride, which increased the pH in intracellular vacuoles from 5.4 to 5.8-6.1, slowed but did not prevent the proteolytic processing and correct localization of pulse-radiolabeled precursors to the lysosomal enzymes alpha-mannosidase and beta-glucosidase. Additionally, ammonium chloride did not affect transport of the enzymes to the Golgi complex, as they acquired resistance to the enzyme endoglycosidase H at the same rate as in control cells. When the pH of lysosomal and endosomal organelles was raised to 6.4 with higher concentrations of ammonium chloride, the percentage of secreted (apparently mis-sorted) precursor polypeptides increased slightly, but proteolytic processing of intermediate forms of lysosomal enzymes to mature forms was greatly reduced. The intermediate and mature forms of alpha-mannosidase and beta-glucosidase did, however, accumulate intracellularly in vesicles similar in density to lysosomes. In contrast, in cells exposed to low concentrations of chloroquine the intravacuolar pH increased only slightly (to 5.7); however, enzymes were inefficiently processed and, instead, rapidly secreted as precursor molecules. Experiments involving the addition of chloroquine at various times during the chase of pulse-radiolabeled cells demonstrated that this weak base acted on a distal Golgi or prelysosomal compartment to prevent the normal sorting of lysosomal enzymes. These results suggest that although acidic endosomal/lysosomal compartments may be important for the complete proteolytic processing of lysosomal enzymes in Dictyostelium, low pH is not essential for the proper targeting of precursor polypeptides. Furthermore, certain amines may induce mis-sorting of these enzymes by pH-independent mechanisms.
采用放射性标记脉冲追踪和亚细胞分级分离程序，分析氯化铵和氯喹这两种弱碱处理的盘基网柄菌细胞中两种溶酶体酶的转运、蛋白水解加工和分选过程。盘基网柄菌缺乏可检测到的不依赖阳离子的甘露糖-6-磷酸受体，是研究溶酶体酶靶向的替代机制的极佳系统。将生长中的细胞暴露于氯化铵中，细胞内液泡的pH值从5.4升高到5.8 - 6.1，这减缓了但并未阻止脉冲放射性标记的溶酶体酶α-甘露糖苷酶和β-葡糖苷酶前体的蛋白水解加工和正确定位。此外，氯化铵并不影响这些酶向高尔基体复合体的转运，因为它们获得对内切糖苷酶H的抗性的速率与对照细胞相同。当用更高浓度的氯化铵将溶酶体和内体细胞器的pH值提高到6.4时，分泌的（明显分选错误的）前体多肽的百分比略有增加，但溶酶体酶中间形式向成熟形式的蛋白水解加工大大减少。然而，α-甘露糖苷酶和β-葡糖苷酶的中间形式和成熟形式确实在细胞内密度与溶酶体相似的囊泡中积累。相反，在暴露于低浓度氯喹的细胞中，液泡内pH值仅略有升高（至5.7）；然而，酶的加工效率低下，而是以前体分子的形式迅速分泌。在脉冲放射性标记细胞的追踪过程中不同时间添加氯喹的实验表明，这种弱碱作用于高尔基体远端或前溶酶体区室，以阻止溶酶体酶的正常分选。这些结果表明，虽然酸性内体/溶酶体区室对于盘基网柄菌中溶酶体酶的完全蛋白水解加工可能很重要，但低pH值对于前体多肽的正确靶向并非必不可少。此外，某些胺可能通过不依赖pH值的机制诱导这些酶的分选错误。
