Derossi D, Joliot A H, Chassaing G, Prochiantz A
Centre National de la Recherche Scientifique Unité de Recherche Associée 1414, Ecole Normale Supérieure, Paris, France.
J Biol Chem. 1994 Apr 8;269(14):10444-50.
The 60-amino acid long homeodomain of Antennapedia crosses biological membranes by an energy-independent mechanism, a phenomenon abolished by directed mutagenesis within the polypeptide C-terminal region. This finding led us to study the internalization of several chemically synthesized peptides derived from the third helix of the homeodomain. We report here that a polypeptide of 16 amino acids in length corresponding to the third helix of the homeodomain deleted of its N-terminal glutamate is still capable of translocating through the membrane. A longer peptide of 20 amino acids also translocates, whereas shorter peptides (15 amino acids) are not internalized by the cells. As is also the case for the entire homeodomain, the 20- and 16-amino acid long peptides are internalized at 4 degrees C, suggesting an energy-independent mechanism of translocation not involving classical endocytosis. The two translocated peptides can be recovered, intact, within the cells, strongly suggesting that they are not targeted to the lysosomal compartment. Finally, substitution of two tryptophans by two phenylalanines strongly diminishes translocation, raising the possibility that the internalization of the third helix is not solely based on its general hydrophobicity.
触角足蛋白60个氨基酸长的同源异型结构域通过一种不依赖能量的机制穿过生物膜，这种现象在多肽C端区域的定向诱变后消失。这一发现促使我们研究源自同源异型结构域第三螺旋的几种化学合成肽的内化过程。我们在此报告，一个对应于同源异型结构域第三螺旋且缺失其N端谷氨酸的16个氨基酸长的多肽仍然能够穿过膜。一个20个氨基酸长的更长肽也能转运，而较短的肽（15个氨基酸）则不会被细胞内化。与整个同源异型结构域的情况一样，20个和16个氨基酸长的肽在4℃时被内化，这表明存在一种不依赖能量的转运机制，不涉及经典的内吞作用。这两种转运的肽可以完整地在细胞内回收，这强烈表明它们不会被靶向到溶酶体区室。最后，用两个苯丙氨酸取代两个色氨酸会大大减少转运，这增加了第三螺旋的内化不仅仅基于其一般疏水性的可能性。
