Sorting of lysosomal membrane glycoproteins lamp-1 and lamp-2 into vesicles distinct from mannose 6-phosphate receptor/γ-adaptin vesicles at the trans-Golgi network
Sorting of lysosomal membrane glycoproteins lamp-1 and lamp-2 into vesicles distinct from mannose 6-phosphate receptor/γ-adaptin vesicles at the trans-Golgi network
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DOI：
10.1074/jbc.273.30.18966
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发表时间：
1998-07-24
影响因子：
4.8
通讯作者：
Carlsson, SR
中科院分区：
文献类型：
--
作者：
Karlsson, K;Carlsson, SR
新合成的溶酶体膜糖蛋白lamp-1和lamp-2主要通过识别其胞质尾中基于酪氨酸的信号序列在高尔基体网络（TGN）处进行分选。目前还不清楚这种信号是如何识别的，以及什么类型的囊泡转运lamp-1和lamp-2。在这里，我们描述了一种方法来产生运输囊泡含有灯蛋白在体外的TGN。该方法基于通过用氚化CMP-唾液酸孵育膜，将放射性唾液酸掺入TGN的糖蛋白中。从标记的膜产生的囊泡需要ATP和胞质溶胶，是温度依赖性和布雷菲德菌素A敏感。Nycodenz梯度分析表明，灯囊泡是不同的囊泡含有γ-适应素和甘露糖B-磷酸受体（MPR），此外，这两种类型的囊泡迁移不同的囊泡含有蛋白质的质膜。灯和MPR被分类成不同的囊泡的结论进一步得到加强的发现，而渥曼青霉素在体外和体内抑制生产的γ-适应素/MPR含有囊泡，这种药物对灯囊泡的产生和灯的排序没有影响。结果表明，含有酪氨酸基序的膜蛋白在TGN分选分离网格蛋白包被的囊泡含有MPR。
Newly synthesized lysosomal membrane glycoproteins lamp-1 and lamp-2 are primarily sorted at the trans-Golgi network (TGN) by recognition of a tyrosine-based signal sequence in their cytoplasmic tails. It is presently unclear how this signal is recognized and what type of vesicle transports lamp-1 and lamp-2. Here, we describe a method to generate transport vesicles containing lamp proteins from the TGN in vitro. The method is based on incorporation of radioactive sialic acid in glycoproteins at the TGN by incubation of membranes with tritiated CMP-sialic acid. The generation of vesicles from labeled membranes required ATP and cytosol, and was temperature-dependent and brefeldin A-sensitive. Analysis on Nycodenz gradients revealed that lamp-vesicles were distinct from vesicles containing gamma-adaptin and mannose B-phosphate receptor (MPR), Moreover, both these types of vesicles migrated differently than vesicles containing proteins destined for the plasma membrane. The conclusion that lamps and MPRs are sorted into different vesicles was further strengthened by the finding that whereas wortmannin both in vitro and in vivo inhibited the production of gamma-adaptin/MPR-containing vesicles, this drug had no effect on the generation of lamp-vesicles and on the sorting of lamps. The results indicate that membrane proteins containing tyrosine-based motifs for sorting at the TGN are segregated from clathrin-coated vesicles containing MPRs.