Dual Anchoring of the GRASP Membrane Tether Promotes trans Pairing
Dual Anchoring of the GRASP Membrane Tether Promotes trans Pairing
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DOI：
10.1074/jbc.m110.116129
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发表时间：
2010-05-21
影响因子：
4.8
通讯作者：
Linstedt, Adam D.
中科院分区：
文献类型：
--
作者：
Bachert, Collin;Linstedt, Adam D.
GRASP蛋白共享一个n端GRASP结构域，介导高尔基池的同型系结，形成延伸的高尔基带。高尔基GM130被认为与GRASP结构域的c端结合，将GRASP65招募到高尔基体上，而稳定的膜结合似乎也依赖于肉豆蔻酰化对N端的锚定。在这里，我们研究了GM130/GRASP65相互作用的性质，并测试了除了膜募集之外，GRASP结构域的双膜接触是否在系结中起作用。发现GM130含有一个c端PDZ配体，该配体结合GRASP65中第二个PDZ样结构域的假定凹槽。为了测试与靶向无关的系聚活性，我们利用了在线粒体膜上进行的系聚实验，其中GRASP膜附着点单独或同时被线粒体靶向跨膜序列取代。只有当C端也锚定时，n端锚定结构体才会锚定；同样，只有当N端被锚定时，c端锚定的构建体才会被锚定。对这种双锚定作用的一种解释是，它定向了GRASP结构域，以防止同一膜内的顺式相互作用，从而有利于相邻膜之间的反式相互作用。事实上，单锚定的GRASP构念虽然在系固中不起作用，但彼此之间相互作用，也束缚和抑制双锚定构念。因此，这项工作阐明了GM130/GRASP65相互作用，并支持一种新的基于取向的膜系系调节模型，其中双膜接触使系系相互作用界面定向，有利于反式而非顺式相互作用。
GRASP proteins share an N-terminal GRASP domain and mediate homotypic tethering of Golgi cisternae to form extended Golgi ribbons. The golgin GM130 is thought to bind the C-terminal side of the GRASP domain to recruit GRASP65 onto the Golgi whereas stable membrane association appears to also depend on anchoring of the N terminus by myristoylation. Here, we examine the nature of the GM130/GRASP65 interaction and test whether the dual membrane contacts of the GRASP domain have a role in tethering beyond membrane recruitment. GM130 was found to contain a C-terminal PDZ ligand that binds the putative groove of the second PDZ-like domain in GRASP65. To test tethering activity independent of targeting, we took advantage of a tethering assay carried out on the mitochondrial membrane in which the GRASP membrane attachment points were individually or simultaneously substituted with mitochondrially targeted transmembrane sequences. N-terminally anchored constructs tethered only if the C terminus was also anchored; and likewise, C-terminally anchored constructs tethered only if the N terminus was anchored. One explanation for the role of this dual anchoring is that it orients the GRASP domain to prevent cis interactions within the same membrane thereby favoring trans interactions between adjacent membranes. Indeed, singly anchored GRASP constructs, although nonfunctional in tethering, interacted with one another and also bound and inhibited dually anchored constructs. This work thus elucidates the GM130/GRASP65 interaction and supports a novel orientation-based model of membrane tether regulation in which dual membrane contact orients the tethering interaction interface to favor trans over cis interactions.