A trs20 Mutation That Mimics an SEDT-Causing Mutation Blocks Selective and Non-Selective Autophagy: A Model for TRAPP III Organization
A trs20 Mutation That Mimics an SEDT-Causing Mutation Blocks Selective and Non-Selective Autophagy: A Model for TRAPP III Organization
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DOI：
10.1111/tra.12095
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发表时间：
2013-10-01
期刊：
影响因子：
4.5
通讯作者：
Sacher, Michael
中科院分区：
文献类型：
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作者：
Brunet, Stephanie;Shahrzad, Nassim;Sacher, Michael
TRapp是一种多亚单位复合体，在膜运输中发挥作用。哺乳动物TRapp蛋白C2的突变与骨骼疾病迟发性脊柱骨盆发育不良(SEDT)有关，这种疾病被认为是由于无法从内质网分泌前胶原而引起的。在这里，我们证明了C2与SNARE蛋白Synaxin 5结合，这种相互作用被SEDT导致的错义突变(D47Y)削弱。有趣的是，酵母C2同源物Trs20p中的等同突变(D46Y)并不阻止顺行运输，但确实影响了内吞作用。Trs20D46Y突变干扰了Trs20p与Trs85p、Trs120p和Trs130p之间的相互作用。大小排除层析表明，这种酵母突变破坏了参与自噬的TRapp III复合体的稳定性。我们进一步表明，该突变既阻止了选择性胞浆到空泡(CVT)途径，也阻止了非选择性自噬。我们证明了TRapp III复合体的表观分子尺寸取决于膜，而TRapp III的存在取决于Atg9p。最后，我们证明了脂化的Bet3p富含在TRapp III中，并且脂化提高了自噬的效率。我们的研究表明，Trs20p作为Trs85p和Trs120p的接头，揭示了TRapp III组装和功能的复杂性。讨论了C2D47Y在SEDT中的意义。
TRAPP is a multisubunit complex that functions in membrane traffic. Mutations in the mammalian TRAPP protein C2 are linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT) that is thought to arise from an inability to secrete procollagen from the endoplasmic reticulum. Here, we show that C2 binds to the SNARE protein Syntaxin 5 and this interaction is weakened by an SEDT-causing missense mutation (D47Y). Interestingly, the equivalent mutation (D46Y) in the yeast C2 homolog Trs20p does not block anterograde traffic but did affect endocytosis. The trs20D46Y mutation interfered with the interaction between Trs20p and Trs85p (TRAPP III-specific subunit), Trs120p and Trs130p (TRAPP II-specific subunits). Size exclusion chromatography suggested that this yeast mutation destabilized the TRAPP III complex that is involved in autophagy. We further show that this mutation blocks both the selective cytosol-to-vacuole (cvt) pathway as well as non-selective autophagy. We demonstrate that the apparent molecular size of the TRAPP III complex is dependent upon membranes, and that the presence of TRAPP III is dependent upon Atg9p. Finally, we demonstrate that lipidated Bet3p is enriched in TRAPP III and that lipidation increases the efficiency of autophagy. Our study suggests that Trs20p acts as an adaptor for Trs85p and Trs120p and reveals complexities in TRAPP III assembly and function. The implications of C2D47Y in SEDT are discussed.