Cardelli J A, Golumbeski G S, Dimond R L
J Cell Biol. 1986 Apr;102(4):1264-70. doi: 10.1083/jcb.102.4.1264.
We are investigating the molecular mechanisms involved in the localization of lysosomal enzymes in Dictyostelium discoideum, an organism that lacks any detectable mannose-6-phosphate receptors. The lysosomal enzymes alpha-mannosidase and beta-glucosidase are both initially synthesized as precursor polypeptides that are proteolytically processed to mature forms and deposited in lysosomes. Time course experiments revealed that 20 min into the chase period, the pulse-labeled alpha-mannosidase precursor (140 kD) begins to be processed, and 35 min into the chase 50% of the polypeptides are cleaved to mature 60 and 58-kD forms. In contrast, the pulse-labeled beta-glucosidase precursor (105 kD) begins to be processed 10 min into the chase period, and by 30 min of the chase all of the precursor has been converted into mature 100-kD subunits. Between 5 and 10% of both precursors escape processing and are rapidly secreted from cells. Endoglycosidase H treatment of immunopurified radioactively labeled alpha-mannosidase and beta-glucosidase precursor polypeptides demonstrated that the beta-glucosidase precursor becomes resistant to enzyme digestion 10 min sooner than the alpha-mannosidase precursor. Moreover, subcellular fractionation studies have revealed that 70-75% of the pulse-labeled beta-glucosidase molecules move from the rough endoplasmic reticulum (RER) to the Golgi complex less than 10 min into the chase. In contrast, 20 min of chase are required before 50% of the pulse-labeled alpha-mannosidase precursor exits the RER. The beta-glucosidase and alpha-mannosidase precursor polypeptides are both membrane associated along the entire transport pathway. After proteolytic cleavage, the mature forms of both enzymes are released into the lumen of lysosomes. These results suggest that beta-glucosidase is transported from the RER to the Golgi complex and ultimately lysosomes at a distinctly faster rate than the alpha-mannosidase precursor. Thus, our results are consistent with the presence of a receptor that recognizes the beta-glucosidase precursor more readily than the alpha-mannosidase precursor and therefore more quickly directs these polypeptides to the Golgi complex.
我们正在研究盘基网柄菌中溶酶体酶定位所涉及的分子机制，盘基网柄菌是一种缺乏任何可检测到的甘露糖-6-磷酸受体的生物体。溶酶体酶α-甘露糖苷酶和β-葡萄糖苷酶最初均以前体多肽的形式合成，这些前体多肽经过蛋白水解加工成为成熟形式，并沉积在溶酶体中。时间进程实验表明，在追踪期开始20分钟后，脉冲标记的α-甘露糖苷酶前体（140kD）开始被加工，追踪35分钟时，50%的多肽被切割成成熟的60kD和58kD形式。相比之下，脉冲标记的β-葡萄糖苷酶前体（105kD）在追踪期开始10分钟后开始被加工，追踪30分钟时，所有前体都已转化为成熟的100kD亚基。两种前体中有5%到10%未被加工，并迅速从细胞中分泌出去。用内切糖苷酶H处理免疫纯化的放射性标记的α-甘露糖苷酶和β-葡萄糖苷酶前体多肽表明，β-葡萄糖苷酶前体比α-甘露糖苷酶前体早10分钟对酶消化产生抗性。此外，亚细胞分级分离研究表明，在追踪期开始不到10分钟时，70%-75%的脉冲标记的β-葡萄糖苷酶分子从粗面内质网（RER）转移到高尔基体复合体。相比之下，在50%的脉冲标记的α-甘露糖苷酶前体离开RER之前，需要20分钟的追踪时间。β-葡萄糖苷酶和α-甘露糖苷酶前体多肽在整个运输途径中都与膜相关。蛋白水解切割后，两种酶的成熟形式被释放到溶酶体腔中。这些结果表明，β-葡萄糖苷酶从RER运输到高尔基体复合体并最终运输到溶酶体的速度明显快于α-甘露糖苷酶前体。因此，我们的结果与存在一种比α-甘露糖苷酶前体更容易识别β-葡萄糖苷酶前体的受体一致，因此能更快地将这些多肽导向高尔基体复合体。
