Ihrke G, Bruns J R, Luzio J P, Weisz O A
Department of Clinical Biochemistry and Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Cambridge CB2 2XY, UK.
EMBO J. 2001 Nov 15;20(22):6256-64. doi: 10.1093/emboj/20.22.6256.
We have examined the trafficking of the mucin-like protein endolyn in transfected, polarized MDCK cells using biochemical approaches and immunofluorescence microscopy. Although endolyn contains a lysosomal targeting motif of the type YXXPhi and was localized primarily to lysosomes at steady state, significant amounts of newly synthesized endolyn were delivered to the apical cell surface. Antibodies to endolyn, but not lamp-2, were preferentially internalized from the apical plasma membrane and efficiently transported to lysosomes. Analysis of endolyn-CD8 chimeras showed that the lumenal domain of endolyn contains apical targeting information that predominates over basolateral information in its cytoplasmic tail. Interestingly, surface polarity of endolyn was independent of O-glycosylation processing, but was reversed by disruption of N-glycosylation using tunicamycin. At all times, endolyn was soluble in cold Triton X-100, suggesting that apical sorting was independent of sphingolipid rafts. Our data indicate that a strong, N-glycan-dependent apical targeting signal in the lumenal domain directs endolyn into a novel biosynthetic pathway to lysosomes, which occurs via the apical surface of polarized epithelial cells.
我们使用生化方法和免疫荧光显微镜检查了转染的极化MDCK细胞中黏蛋白样蛋白内多琳的运输情况。尽管内多琳含有YXXPhi类型的溶酶体靶向基序，并且在稳态时主要定位于溶酶体，但大量新合成的内多琳被递送至顶端细胞表面。针对内多琳而非lamp-2的抗体优先从顶端质膜内化，并有效地转运至溶酶体。对内多琳-CD8嵌合体的分析表明，内多琳的腔结构域包含顶端靶向信息，该信息在其细胞质尾中比基底外侧信息占主导地位。有趣的是，内多琳的表面极性与O-糖基化加工无关，但使用衣霉素破坏N-糖基化可使其反转。在所有时候，内多琳在冷的Triton X-100中均呈可溶状态，这表明顶端分选与鞘脂筏无关。我们的数据表明，腔结构域中一个强大的、依赖N-聚糖的顶端靶向信号将内多琳导向一条新的溶酶体生物合成途径，该途径通过极化上皮细胞的顶端表面发生。
