Hansen S H, Sandvig K, van Deurs B
Department of Anatomy, Panum Institute, University of Copenhagen, Denmark.
J Cell Biol. 1993 Oct;123(1):89-97. doi: 10.1083/jcb.123.1.89.
We have previously demonstrated that the preendosomal compartment in addition to clathrin-coated vesicles, comprises distinct nonclathrin coated endocytic vesicles mediating clathrin-independent endocytosis (Hansen, S. H., K. Sandvig, and B. van Deurs. 1991. J. Cell Biol. 113:731-741). Using K+ depletion in HEp-2 cells to block clathrin-dependent but not clathrin-independent endocytosis, we have now traced the intracellular routing of these nonclathrin coated vesicles to see whether molecules internalized by clathrin-independent endocytosis are delivered to a unique compartment or whether they reach the same early and late endosomes as encountered by molecules internalized with high efficiency through clathrin-coated pits and vesicles. We find that Con A-gold internalized by clathrin-independent endocytosis is delivered to endosomes containing transferrin receptors. After incubation of K(+)-depleted cells with Con A-gold for 15 min, approximately 75% of Con A-gold in endosomes is colocalized with transferrin receptors. Endosomes containing only Con A-gold may be accounted for either by depletion of existing endosomes for transferrin receptors or by de novo generation of endosomes. Cationized gold and BSA-gold internalized in K(+)-depleted cells are also delivered to endosomes containing transferrin receptors. h-lamp-1-enriched compartments are only reached occasionally within 30 min in K(+)-depleted as well as in control cells. Thus, preendosomal vesicles generated by clathrin-independent endocytosis do not fuse to any marked degree with late endocytic compartments. These data show that in HEp-2 cells, molecules endocytosed without clathrin are delivered to the same endosomes as reached by transferrin receptors internalized through clathrin-coated pits.
我们之前已经证明，除网格蛋白包被小泡外，前内体区室还包含介导网格蛋白非依赖性内吞作用的独特的非网格蛋白包被的内吞小泡（Hansen, S. H., K. Sandvig, and B. van Deurs. 1991. J. Cell Biol. 113:731 - 741）。利用HEp - 2细胞中的钾离子耗竭来阻断网格蛋白依赖性而非网格蛋白非依赖性内吞作用，我们现在追踪了这些非网格蛋白包被小泡的细胞内路径，以查看通过网格蛋白非依赖性内吞作用内化的分子是被递送到一个独特的区室，还是它们会到达与通过网格蛋白包被小窝和小泡高效内化的分子所遇到的相同的早期和晚期内体。我们发现，通过网格蛋白非依赖性内吞作用内化的伴刀豆球蛋白A - 金被递送到含有转铁蛋白受体的内体。在用伴刀豆球蛋白A - 金孵育钾离子耗竭的细胞15分钟后，内体中约75%的伴刀豆球蛋白A - 金与转铁蛋白受体共定位。仅含有伴刀豆球蛋白A - 金的内体可能是由于现有转铁蛋白受体内体的耗尽，或者是由于内体的从头生成。在钾离子耗竭的细胞中内化的阳离子化金和牛血清白蛋白 - 金也被递送到含有转铁蛋白受体的内体。在钾离子耗竭的细胞以及对照细胞中，富含溶酶体相关膜蛋白1（h - lamp - 1）的区室在30分钟内只是偶尔会被到达。因此，由网格蛋白非依赖性内吞作用产生的前内体小泡与晚期内吞区室没有明显程度的融合。这些数据表明，在HEp - 2细胞中，无网格蛋白内吞的分子被递送到与通过网格蛋白包被小窝内化的转铁蛋白受体所到达的相同的内体。
