Wessling-Resnick M, Braell W A
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1990 Oct 5;265(28):16751-9.
A cell-free assay to monitor receptor-mediated endocytic processes has been developed that uses biotinylated transferrin and avidin-linked beta-galactosidase as receptor-associated and fluid-phase probes, respectively (Wessling-Resnick, M., and Braell, W. A. (1990) J. Biol. Chem. 265, 690-699). The fusion of vesicles from heterologous sources can be detected in this assay: endocytic vesicles from K562 cells (a human cell line) will fuse with vesicles from Chinese hamster ovary cells. Fusion between endocytic vesicles is inhibited upon treatment with N-ethylmaleimide but can be restored by the addition of untreated cytosol from either cell type. The in vitro fusion reaction is also inhibited by the nonhydrolyzable nucleotide analogs guanosine 5'-(3-thiotriphosphate) (GTP gamma S) and adenosine 5'-(3-thiotriphosphate) (ATP gamma S). Other nonhydrolyzable guanine nucleotides are found to inhibit the in vitro reaction in the following order of potency: GTP gamma S greater than 5'-guanylyl imidodiphosphate (GTP-PNP) greater than alpha,beta-methylene GTP (GTP-PCP). The inhibitory effects of the nonhydrolyzable analogs of GTP and ATP are not additive. Moreover, excess GTP relieves the inhibition by GTP gamma S more than it relieves the inhibition by ATP gamma S, while excess ATP preferentially alleviates ATP gamma S (not GTP gamma S) inhibition. These properties suggest that the two nucleotides exert their effects at distinct points in the fusion process. Although micromolar levels of excess Ca2+ also inhibit vesicle fusion, the inhibition exerted by GTP gamma S appears to proceed via a pathway independent of the divalent cation. The GTP gamma S-sensitive step in endocytic vesicle fusion is found to occur at a mechanistic stage prior to and distinct from the N-ethylmaleimide-sensitive step of the reaction. This situation permits the accumulation of a membrane vesicle intermediate in the presence of GTP gamma S; subsequent incubation of these vesicles with cytosol and GTP restores their fusion competence. Characteristics of in vitro endocytic vesicle fusion suggest that similarities exist with steps of the fusion mechanism involved with membrane traffic events of the secretory pathway.
已开发出一种用于监测受体介导的内吞过程的无细胞测定法，该方法分别使用生物素化转铁蛋白和抗生物素蛋白连接的β-半乳糖苷酶作为受体相关探针和液相探针（韦斯林 - 雷斯尼克，M.，和布雷尔，W. A.（1990年）《生物化学杂志》265卷，690 - 699页）。在此测定法中可检测到来自异源的囊泡融合：K562细胞（一种人类细胞系）的内吞囊泡将与中国仓鼠卵巢细胞的囊泡融合。用N - 乙基马来酰亚胺处理后，内吞囊泡之间的融合受到抑制，但通过添加来自任一细胞类型的未处理细胞溶胶可恢复融合。体外融合反应也受到不可水解的核苷酸类似物鸟苷5'-（3 - 硫代三磷酸）（GTPγS）和腺苷5'-（3 - 硫代三磷酸）（ATPγS）的抑制。发现其他不可水解的鸟嘌呤核苷酸按以下效力顺序抑制体外反应：GTPγS大于5'-鸟苷酰亚胺二磷酸（GTP - PNP）大于α，β-亚甲基GTP（GTP - PCP）。GTP和ATP的不可水解类似物的抑制作用不是相加的。此外，过量的GTP比减轻ATPγS的抑制更能减轻GTPγS的抑制，而过量的ATP优先减轻ATPγS（而非GTPγS）的抑制。这些特性表明这两种核苷酸在融合过程的不同点发挥作用。尽管微摩尔水平的过量Ca2 +也抑制囊泡融合，但GTPγS施加的抑制似乎通过一条独立于二价阳离子的途径进行。发现内吞囊泡融合中对GTPγS敏感的步骤发生在反应的N - 乙基马来酰亚胺敏感步骤之前且与之不同的机制阶段。这种情况允许在存在GTPγS的情况下积累膜囊泡中间体；随后将这些囊泡与细胞溶胶和GTP一起孵育可恢复其融合能力。体外内吞囊泡融合的特性表明，其与分泌途径膜运输事件的融合机制步骤存在相似性。
