Bush J M, Cardelli J A
Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport 71130.
J Biol Chem. 1989 May 5;264(13):7630-6.
To explain the different secretion kinetics of lysosomal enzymes in Dictyostelium discoideum, previous investigators have hypothesized the existence of a heterogeneous population of lysosomes containing either the enzyme acid phosphatase or other hydrolase enzymes. This proposal predicts that at least two targeting mechanisms exist for lysosomal enzymes in this organism. To begin to investigate this possibility, the transport, processing, and targeting of acid phosphatase was studied by using a combination of radiolabel pulse-chase procedures, subcellular fractionations, and indirect immunofluorescence microscopy. Acid phosphatase was initially synthesized in axenically growing cells as a 56-kDa precursor polypeptide that was proteolytically processed after 20 min to a 55-kDa mature protein. This enzyme was rapidly transported from the endoplasmic reticulum to Golgi complex (halftime of 3 min) as measured by the acquisition of resistance to the enzyme endoglycosidase H. Furthermore, Percoll gradient fractionations indicated that radiolabeled forms of acid phosphatase reached dense lysosomal vesicles at about the same time as final processing was occurring. Proper sorting of acid phosphatase in D. discoideum apparently was not critically dependent on low intravacuolar pH since the addition of ammonium chloride did not stimulate the missorting and secretion of acid phosphatase. These results are very similar to previous observations concerning other Dictyostelium lysosomal enzymes. Consistent with the existence of a heterogeneus population of lysosomes, the percentage of radiolabeled acid phosphatase secreted 4 h into a chase period was 15-fold lower as compared with another lysosomal enzyme, beta-glucosidase. However, acid phosphatase, alpha-mannosidase, and beta-glucosidase were all predominantly colocalized as determined by indirect immunofluorescence, which for the first time demonstrates the homogeneous nature of the lysosomal system in D. discoideum. Taken together these results suggest that the processing and transport of acid phosphatase may be similar in nature to the glycosidases. However, the different kinetics of secretion of acid phosphatase versus the colocalized glycosidase enzymes suggests that an undefined mechanism operates to distinguish these classes of enzymes at a step after localization to lysosomes but prior to secretion.
为了解释盘基网柄菌中溶酶体酶不同的分泌动力学，之前的研究人员推测存在含有酸性磷酸酶或其他水解酶的溶酶体异质群体。这一推测表明，该生物体中溶酶体酶至少存在两种靶向机制。为了开始研究这种可能性，通过结合放射性标记脉冲追踪程序、亚细胞分级分离和间接免疫荧光显微镜技术，对酸性磷酸酶的转运、加工和靶向进行了研究。酸性磷酸酶最初在无菌培养的细胞中作为一种56 kDa的前体多肽合成，20分钟后经蛋白水解加工成为55 kDa的成熟蛋白。通过获得对内切糖苷酶H的抗性来测量，该酶从内质网快速转运至高尔基体复合体（半衰期为3分钟）。此外，Percoll梯度分级分离表明，放射性标记的酸性磷酸酶形式在最终加工发生时大约同时到达致密的溶酶体囊泡。盘基网柄菌中酸性磷酸酶的正确分选显然并不严格依赖于液泡内的低pH值，因为添加氯化铵并不会刺激酸性磷酸酶的错误分选和分泌。这些结果与之前关于其他盘基网柄菌溶酶体酶的观察结果非常相似。与溶酶体异质群体的存在一致，在追踪期4小时分泌的放射性标记酸性磷酸酶的百分比与另一种溶酶体酶β-葡萄糖苷酶相比低15倍。然而，通过间接免疫荧光测定，酸性磷酸酶、α-甘露糖苷酶和β-葡萄糖苷酶主要都共定位，这首次证明了盘基网柄菌中溶酶体系统的同质性。综合这些结果表明，酸性磷酸酶的加工和转运在性质上可能与糖苷酶相似。然而，酸性磷酸酶与共定位的糖苷酶不同的分泌动力学表明，在定位到溶酶体后但在分泌之前的某个步骤中，存在一种未明确的机制来区分这些酶类。
