Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intracellular trafficking.
Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intracellular trafficking.
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DOI：
10.1002/jcb.10112.abs
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发表时间：
2002
影响因子：
4
通讯作者：
E. Kalinina;O. Varlamov;L. Fricker
中科院分区：
文献类型：
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作者：
E. Kalinina;O. Varlamov;L. Fricker
金属羧肽酶D（CPD）是一种1型跨膜蛋白，其在通过分泌途径的蛋白质的加工中起作用。以前，CPD被发现是丰富的反式高尔基体网络（TGN）和循环之间的这间室和细胞表面。在本研究中，在AtT-20细胞系中研究了CPD胞质尾部特定区域在细胞内运输中的作用。当CPD跨膜区和胞质尾部连接到白蛋白的C-末端时，该蛋白质保留在TGN中并循环到细胞表面。缺失分析表明，一个C-末端区域的功能在TGN保留;从C-末端去除10个氨基酸的融合蛋白的量大大增加，进入新生囊泡，芽从高尔基体，但不影响的融合蛋白的半衰期或细胞表面蛋白的能力，返回到TGN。由于10个残基的缺失破坏了酪蛋白激酶2（CK 2）共有位点，因此该位点（TDT）中的两个Thr突变为Ala（ADA）或Glu（EDE）。这两种突变都没有增加从TGN出芽的速率，尽管ADA突变体的半衰期比野生型序列或EDE突变体短。衔接蛋白-1和-2结合到大多数的缺失突变体，EDE点突变体，和CK 2-磷酸化的CPD尾，但不野生型尾。两者合计，这些结果表明，CPD本地化的TGN需要静态保留涉及的C-末端结构域和磷酸化的CK 2位点，调节衔接蛋白的结合。
Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intracellular trafficking were investigated in the AtT-20 cell line. When the CPD transmembrane region and cytosolic tail are attached to the C-terminus of albumin, this protein is retained in the TGN and cycles to the cell surface. Deletion analysis indicates that a C-terminal region functions in TGN-retention; removal of 10 amino acids from the C-terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half-life of the fusion protein or the ability of cell surface protein to return to the TGN. Because the 10-residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half-life than either the wild type sequence or the EDE mutant. Adaptor protein-1 and -2 bind to most of the deletion mutants, the EDE point mutant, and the CK2-phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C-terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins.