Specificity of the GET pathway for TA protein insertion in Arabidopsis thaliana

尾部锚定蛋白(GET)途径的引导进入被认为是TA蛋白插入内质网膜的典型例子。胞浆ATPase ScGET3将新生的TA蛋白穿梭到ER受体ScGET1/2上进行膜插入。虽然生化和结构分析已经建立了这一看似令人信服的模型，但关于这一机制的生理学和遗传学证据很少。在酵母中，GET功能的丧失是可有可无的，到目前为止，只有有限数量的TA蛋白被测试依赖于GET进行膜插入，这留下了一个悬而未决的问题，即哪个替代途径(S)可能负责大多数TA蛋白的翻译后插入。ScGET3还在蛋白质质量控制中扮演分子伴侣的角色，与错误折叠的TA蛋白结合，并将这些蛋白恢复到插入能力状态或针对它们进行降解。我们已经确定并表征了植物中可能的GET途径的主要同源物，并发现在拟南芥中，GET功能丧失的品系显示出较短的根毛，而其他情况下生长正常。虽然这种缺陷至少部分可以归因于一个重要的质膜陷阱(SYP123)丰度的减少，但缺乏更明显的表型和对不与ATGET同源基因结合的TA蛋白的鉴定表明存在替代的插入途径。此外，细胞质AtGET3a在Atget1系中的异常表达导致了一系列严重的表型，从幼苗死亡到生长和育性下降。我们的目标是了解AtGET3a针对内质网的TA蛋白的特异性，以及它执行的替代细胞功能(如果有的话)。此外，我们还试图确定TA蛋白插入的内质网膜上的关键角色，并确定参与替代插入途径的蛋白质(S)。

The Guided Entry of Tail-anchored proteins (GET) pathway is viewed as textbook example of TA protein insertion into the ER membrane. The cytosolic ATPase ScGET3 shuttles nascent TA proteins to the ER receptors ScGET1/2 for membrane insertion. While biochemical and structural analyses have established this seemingly compelling model, physiological and genetic evidence for the mechanism is scarce. In yeast, loss of GET function is dispensable and only a limited number of TA proteins have so far been tested to depend on GET for membrane insertion, leaving the question unresolved which alternative pathway(s) is(are) likely responsible for posttranslational insertion of most TA proteins. ScGET3 also acts as a molecular chaperone in protein quality control, binding to misfolded TA proteins and either reverting these to an insertion competent state or targeting them for degradation.We have identified and characterised the main orthologues of a putative GET pathway in plants and uncovered that GET loss-of-function lines in Arabidopsis show shorter root hairs, while otherwise growing normally. While this defect can at least in part be attributed to reduced abundance of an important plasma membrane SNARE (SYP123), lack of a more pronounced phenotype and identification of TA proteins that do not bind to AtGET orthologues suggest existence of alternative insertion pathways. Moreover, aberrant expression of the cytosolic AtGET3a in the Atget1 line leads to a range of severe phenotypes from seedling lethality to reduced growth and fertility. We aim to understand the specificity of AtGET3a in targeting TA proteins to the ER and what, if any, alternative cellular functions does it execute. Also, we seek to determine the key players in the ER membrane for TA protein insertion and identify protein(s) that engage in an alternative insertion pathway.