Schneider N, Schwartz J M, Köhler J, Becker M, Schwarz H, Gerisch G
Max-Planck-lnstitut für Biochemie, Martinsried, Germany.
Biol Cell. 2000 Oct;92(7):495-511. doi: 10.1016/s0248-4900(00)01102-3.
Golvesin is a new protein associated with membranes of the Golgi apparatus and post-Golgi vesicles in Dictyostelium cells. An internal hydrophobic sequence of 24 amino-acid residues is responsible for anchoring golvesin to the membranes of these organelles. In an attempt to visualize organelle dynamics in vivo, we have used specific antibody and other labels to localize golvesin-green fluorescent protein (GFP) constructs to different cellular compartments. With a GFP tag at its N-terminus, golvesin shows the same localization as the untagged protein. It is transferred to two post-Golgi compartments, the endosomal and contractile vacuole systems. Endosomes are decorated with GFP-golvesin within less than 10 min of their internalisation, and keep the label during the acidic phase of the pathway. Blockage of the C-terminus with GFP causes entrapment of the protein in the Golgi apparatus, indicating that a free C-terminus is required for transfer of golvesin to any of the post-Golgi compartments. The C-terminally tagged golvesin proved to be a reliable Golgi marker in Dictyostelium cells revealing protrusion of Golgi tubules at peak velocities of 3 to 4 microm x s(-1). The fusion protein is retained in Golgi vesicles during mitosis, visualizing Golgi disassembly and reorganization in line with cytokinesis.
高尔维辛是一种与盘基网柄菌细胞中高尔基体和高尔基体后囊泡膜相关的新蛋白质。一段由24个氨基酸残基组成的内部疏水序列负责将高尔维辛锚定到这些细胞器的膜上。为了在体内观察细胞器的动态变化，我们使用了特异性抗体和其他标记物，将高尔维辛-绿色荧光蛋白（GFP）构建体定位到不同的细胞区室。在其N端带有GFP标签时，高尔维辛显示出与未标记蛋白相同的定位。它被转运到两个高尔基体后区室，即内体和收缩泡系统。内体在其内化后不到10分钟内就被GFP-高尔维辛标记，并在该途径的酸性阶段保留该标记。用GFP阻断C端会导致该蛋白被困在高尔基体中，这表明高尔维辛转移到任何高尔基体后区室都需要一个游离的C端。在盘基网柄菌细胞中，C端标记的高尔维辛被证明是一种可靠的高尔基体标记物，可显示高尔基体小管以3至4微米×秒（-1）的峰值速度突出。在有丝分裂期间，融合蛋白保留在高尔基体囊泡中，显示高尔基体随着胞质分裂而解体和重组。
