Naim H Y, Dodds D T, Brewer C B, Roth M G
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235-9038, USA.
J Cell Biol. 1995 Jun;129(5):1241-50. doi: 10.1083/jcb.129.5.1241.
In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells. The rates of internalization of HA-Y543 and HA-Y543,R546 from the basolateral surface of polarized MDCK cells resembled those in nonpolarized cells, whereas their rates of internalization from the apical cell surface were fivefold slower. However, HA-Y543,R546 was internalized approximately threefold faster than HA-Y543 at both membrane domains, indicating that apical endocytic pits in polarized MDCK cells retained the ability to discriminate between different internalization signals. Slower internalization from the apical surface could not be explained by a limiting number of coated pits: apical membrane contained 0.7 as many coated pits per cell cross-section as did basolateral membranes. 10-14% of HA-Y543 at the apical surface of polarized MDCK cells was found in coated pits, a percentage not significantly different from that observed in apical coated pits of nonpolarized MDCK cells, where internalization was fivefold faster. Thus, there was no lack of binding sites for HA-Y543 in apical coated pits of polarized cells. However, at the apical surface many more shallow pits, and fewer deep, mature pits, were observed than were seen at the basolateral. These results suggest that the slower internalization at the apical surface is due to slower maturation of coated pits, and not to a difference in recognition of internalization signals.
在极化的上皮MDCK细胞中，所有已知的内源性内吞受体都位于基底外侧结构域。通常被分选到顶端质膜的流感病毒血凝素（HA），可通过其短细胞质结构域中的特定突变转化为基底外侧蛋白，这些突变还会产生内化信号。对于其中一些突变，分选到基底外侧表面并不完全，使得可以在MDCK细胞的顶端和基底外侧表面比较两种仅因内化信号的单个氨基酸不同的蛋白的内化情况。极化MDCK细胞基底外侧表面的HA-Y543和HA-Y543,R546的内化速率与非极化细胞中的相似，而它们从顶端细胞表面的内化速率则慢五倍。然而，在两个膜结构域中，HA-Y543,R546的内化速度比HA-Y543快约三倍，这表明极化MDCK细胞中的顶端内吞小窝保留了区分不同内化信号的能力。顶端表面内化较慢不能用被膜小窝数量有限来解释：顶端膜每细胞横截面所含的被膜小窝数量是基底外侧膜的0.7倍。在极化MDCK细胞顶端表面，10 - 14%的HA-Y543存在于被膜小窝中，这一百分比与内化速度快五倍的非极化MDCK细胞顶端被膜小窝中的百分比没有显著差异。因此，极化细胞顶端被膜小窝中不存在HA-Y543的结合位点缺失情况。然而，在顶端表面观察到的浅小窝比基底外侧的多得多，而深的、成熟的小窝则比基底外侧的少。这些结果表明，顶端表面内化较慢是由于被膜小窝成熟较慢，而不是内化信号识别的差异。
