Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway
Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway
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DOI：
10.1074/jbc.m109.026435
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发表时间：
2009-11-06
影响因子：
4.8
通讯作者：
Kambe, Taiho
中科院分区：
文献类型：
--
作者：
Fukunaka, Ayako;Suzuki, Tomoyuki;Kambe, Taiho
大多数CDF/ZNT锌转运蛋白形成均一低聚物。然而，在早期的分泌途径中，ZnT5、ZnT6和它们的同源异构体形成杂寡体，它们将锌装载到需要锌的酶上，维持分泌途径的功能。这种异位齐聚的细节仍有待阐明，而在其他CDF/ZNT家族蛋白中发生的同位齐聚也有更多的信息。在这里，我们通过免疫共沉淀实验、诱变和嵌合体研究来解决这个问题，这些实验是在缺乏锌锌蛋白的鸡DT40细胞中进行的，包括诱变和嵌合体研究。我们发现hZnT5和hZnT6结合形成杂二聚体，但不形成比杂二聚体更大的络合物。对hZnT6的诱变实验表明，CDF/ZNT蛋白在跨膜结构域II和V上保守的亲水性氨基酸残基与锌结合不参与，但在其他CDF/ZNT家族低聚物中锌的转运是必需的。我们还发现，hZnT5的N-末端不是与hZnT6功能相互作用所必需的，而胞浆中的C-末端对于确定hZnT6是形成异源二聚体的配对分子是重要的。在DT40细胞中，在内质网应激期内源性诱导了缺失N-末端半部分的cZnT5变异体，因此在这种情况下似乎有向需锌酶供应锌的功能。本文概述的结果提供了有关CDF/ZNT蛋白异源二聚作用的新信息，CDF/ZNT蛋白在早期分泌途径中发挥作用。
The majority of CDF/ZnT zinc transporters form homooligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.