Fuhrer C, Geffen I, Spiess M
Department of Biochemistry, University of Basel, Switzerland.
J Cell Biol. 1991 Aug;114(3):423-31. doi: 10.1083/jcb.114.3.423.
The clustering of plasma membrane receptors in clathrin-coated pits depends on determinants within their cytoplasmic domains. In several cases, individual tyrosine residues were shown to be necessary for rapid internalization. We have mutated the single tyrosine at position 5 in the cytoplasmic domain of the major subunit H1 of the asialoglycoprotein receptor to alanine. Expressed in fibroblasts cells, the mutant protein was accumulated in the plasma membrane, and its rate of internalization was reduced by a factor of four. The residual rate of endocytosis, however, was still significantly higher than that of resident plasma membrane proteins. Upon acidification of the cytoplasm, which specifically inhibits the formation of clathrin-coated vesicles but not uptake of the fluid phase marker Lucifer yellow, residual endocytosis was blocked. By immunoelectron microscopy mutant H1 could be directly demonstrated in coated pits. The fraction of wild-type and mutant H1 present in coated pits as determined by immunogold localization correlated well with the respective rates of internalization. Thus, mutation of tyrosine-5 only partially inactivates recognition of H1 for incorporation into coated pits.
质膜受体在网格蛋白包被小窝中的聚集取决于其胞质结构域内的决定因素。在几种情况下，单个酪氨酸残基被证明是快速内化所必需的。我们已将去唾液酸糖蛋白受体主要亚基H1胞质结构域中第5位的单个酪氨酸突变为丙氨酸。在成纤维细胞中表达时，突变蛋白积聚在质膜中，其内化速率降低了四倍。然而，残余的内吞速率仍显著高于驻留质膜蛋白的内吞速率。当细胞质酸化时，这会特异性抑制网格蛋白包被小泡的形成，但不会抑制液相标记物路西法黄的摄取，残余的内吞作用被阻断。通过免疫电子显微镜可直接在包被小窝中检测到突变型H1。通过免疫金定位确定的野生型和突变型H1存在于包被小窝中的比例与各自的内化速率密切相关。因此，酪氨酸-5的突变仅部分失活了H1被纳入包被小窝的识别过程。
