Mutagenesis and structural modeling implicate RME-8 IWN domains as conformational control points.
Mutagenesis and structural modeling implicate RME-8 IWN domains as conformational control points.
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DOI：
10.1371/journal.pgen.1010296
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发表时间：
2022-10
期刊：
影响因子：
4.5
通讯作者：
中科院分区：
文献类型：
--
作者：
内吞作用后，跨膜货物被不同地分类到降解或回收途径。这一过程是通过在单个核内体的限制膜上招募到物理上不同的降解或循环微域而促进的。运输所需的内体分选复合物（ESCRT）标记降解微域，而再循环域由反转录复合物和相关蛋白RME-8和SNX-1标记。内体微域的分离也由RME-8和SNX-1控制，至少部分是通过从循环微域去除降解成分HRS/HGRS-1来实现的。这种活性可能是由于RME-8 DNAJ结构域在核内体上募集和激活伴侣Hsc70。为了更好地了解RME-8的功能机制，我们对RME-8进行了新的系统发育分析，并发现了新的保守序列特征。在一种互补的方法中，我们进行了结构-功能分析，确定了c端对微域定位和可能的底物结合很重要，而n端序列超出已知的单个n端ph样结构域对内体募集很重要。随机诱变发现IWN4和IWN3对自身抑制DNAJ结构域结合很重要，IWN3在HRS脱膜活性中起关键作用。结合AlphaFold结构预测和RME-8的体内突变分析，我们提出了一个模型，其中SNX-1和IWN结构域控制RME-8的构象，从而控制DNAJ结构域的生产暴露。此外，我们提出RME-8的激活是周期性的，SNX-1作为RME-8脱膜活性的激活剂和靶标。真核生物的细胞，包括动物、植物和真菌，含有几个特殊的膜结合的隔室，它们执行生命所必需的功能。膜蛋白在这些隔室之间的转运是一个活跃的研究领域，在人类代谢和神经疾病中具有重要意义。内体是一种重要的隔室，从细胞外膜吸收的跨膜蛋白质在这里被分类降解或再利用。这种分类部分是通过物理上不同的蛋白质外壳，降解和回收微域实现的。关于这些微域是如何相互分离的，我们知之甚少。在这项工作中，我们揭示了回收蛋白RME-8是如何工作的，RME-8是微结构域分离的关键参与者。我们报道RME-8可能在内体膜上被同质寡聚化抑制，直到它被伴生循环蛋白SNX-1激活。这种激活允许RME-8剥离降解分选蛋白以限制微结构域和其自身的激活剂SNX-1作为负反馈调节的手段。然而，RME-8和SNX-1并不是单独行动的，而是一个庞大的回收机器的一部分，要在更广泛的背景下揭示它们的功能，还有许多令人兴奋的工作要做。
After endocytosis, transmembrane cargo is differentially sorted into degradative or recycling pathways. This process is facilitated by recruitment into physically distinct degradative or recycling microdomains on the limiting membrane of individual endosomes. Endosomal sorting complexes required for transport (ESCRT) mark the degradative microdomain, while the recycling domain is marked by the retromer complex and associated proteins RME-8 and SNX-1. The separation of endosomal microdomains is also controlled by RME-8 and SNX-1, at least in part via removal of degradative component HRS/HGRS-1 from the recycling microdomain. This activity is likely due to recruitment and activation of chaperone Hsc70 on the endosome by the RME-8 DNAJ domain. To better understand the mechanism of RME-8 function we performed a new phylogenetic analysis of RME-8 and identified new conserved sequence features. In a complementary approach, we performed structure-function analysis that identified the C-terminus as important for microdomain localization and likely substrate binding, while N-terminal sequences beyond the known single N-terminal PH-like domain are important for endosome recruitment. Random mutagenesis identified IWN4, and by analogy IWN3, to be important for the autoinhibitory DNAJ domain binding, with IWN3 playing a critical role in HRS uncoating activity. Combining AlphaFold structural predictions with in vivo mutation analysis of RME-8, we propose a model whereby SNX-1 and the IWN domains control the conformation of RME-8 and hence the productive exposure of the DNAJ domain. Furthermore, we propose that the activation of RME-8 is cyclical, with SNX-1 acting as an activator and a target of RME-8 uncoating activity. The cells of eukaryotic organisms, including animals, plants, and fungi contain several specialized membrane-bound compartments which perform functions essential for life. Trafficking of membrane proteins between these compartments is an active area of study with important implications in human metabolic and neurologic disease. Endosomes are remarkable compartments where transmembrane proteins taken in from the cell’s outer membrane are sorted for degradation or reuse. This sorting is in part achieved by physically distinct protein coats, the degradative and recycling microdomains. Little about how these microdomains segregate from each other is known. In this work, we reveal how recycling protein RME-8, a key player in microdomain segregation, works. We report that RME-8 is likely inhibited on endosomal membranes by homo-oligomerization until it is activated by companion recycling protein SNX-1. This activation allows RME-8 to un-coat both degradative sorting proteins to limit of the microdomains and its own activator SNX-1 as a means of negative feedback regulation. RME-8 and SNX-1 do not act alone, however, but are parts of an expansive recycling machinery, with much exciting work yet to be done to uncover their functions within this broader context.
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影响因子：
3.5
作者：
Girard, Martine;McPherson, Peter S.
通讯作者：
McPherson, Peter S.
影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
3.7
作者：
Kim E;Sun L;Gabel CV;Fang-Yen C
通讯作者：
Fang-Yen C
DOI：
10.1083/jcb.200311084
发表时间：
2004-03-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Chang HC;Hull M;Mellman I
通讯作者：
Mellman I
影响因子：
4
作者：
Seaman, Matthew N. J.
通讯作者：
Seaman, Matthew N. J.