Specific Disruption of Ras2 CAAX Proteolysis Alters Its Localization and Function.
Specific Disruption of Ras2 CAAX Proteolysis Alters Its Localization and Function.
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DOI：
10.1128/spectrum.02692-22
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发表时间：
2023-02-14
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
--
作者：
许多CAAX蛋白，如Ras GT3，在其羧基末端经历一系列翻译后修饰（即，半胱氨酸异戊二烯化、AAX的内蛋白水解和羧基甲基化）。然而，一些CAAX蛋白仅经历异戊烯基化修饰，例如酿酒酵母Hsp 40 Ydj 1。我们以前观察到，改变Ydj 1的CAAX基序从异戊烯化，只有规范导致改变Ydj 1的功能和定位。在这里，我们研究了相互变化的影响，改变了典型的CAAX基序的S。cerevisiae Ras 2转化为仅异戊二烯化。我们观察到CAAX基序的类型影响Ras 2蛋白水平、定位和功能。此外，我们观察到，使用异戊烯基化的序列阶段过度活跃的Ras 2-G19 V作为法尼基化和非蛋白水解的中间体，导致不同的表型相对于分期的遗传RCE 1缺失策略，同时影响许多CAAX蛋白。这些研究结果表明，一个异戊二烯化的CAAX基序是有用的探测条件下，其他CAAX蛋白质通常被修改的CAAX蛋白水解对Ras 2的具体影响。我们建议，我们的策略可以很容易地应用于广泛的CAAX蛋白质，以检查CAAX蛋白水解对其功能的具体影响。重要提示CAAX蛋白会受到多种翻译后修饰：半胱氨酸异戊烯化、CAAX蛋白水解和羧基甲基化。为了研究CAAX蛋白水解，本研究采用了新的方法，使用抗蛋白水解的CAAX序列将酿酒酵母Ras 2 GTdR分期为法尼基化和非蛋白水解状态。我们的方法特别限制了破坏CAAX蛋白水解对Ras 2的影响。这代表了对先前方法的改进，其中CAAX蛋白水解通过基因敲除、小干扰RNA敲除或Rce 1 CAAX蛋白酶的生化抑制来抑制，这可能导致由于Rce 1对多种CAAX蛋白的作用而导致的多效性和不清楚的效应归属。我们的方法产生的结果证明了CAAX蛋白水解对Ras 2的功能，定位和其他特性的特定影响，突出了这种方法在其他蛋白质背景下研究CAAX蛋白水解的影响的实用性。
Many CAAX proteins, such as Ras GTPase, undergo a series of posttranslational modifications at their carboxyl terminus (i.e., cysteine prenylation, endoproteolysis of AAX, and carboxylmethylation). Some CAAX proteins, however, undergo prenylation-only modification, such as Saccharomyces cerevisiae Hsp40 Ydj1. We previously observed that altering the CAAX motif of Ydj1 from prenylation-only to canonical resulted in altered Ydj1 function and localization. Here, we investigated the effects of a reciprocal change that altered the well-characterized canonical CAAX motif of S. cerevisiae Ras2 to prenylation-only. We observed that the type of CAAX motif impacted Ras2 protein levels, localization, and function. Moreover, we observed that using a prenylation-only sequence to stage hyperactive Ras2-G19V as a farnesylated and nonproteolyzed intermediate resulted in a different phenotype relative to staging by a genetic RCE1 deletion strategy that simultaneously affected many CAAX proteins. These findings suggested that a prenylation-only CAAX motif is useful for probing the specific impact of CAAX proteolysis on Ras2 under conditions where other CAAX proteins are normally modified. We propose that our strategy could be easily applied to a wide range of CAAX proteins for examining the specific impact of CAAX proteolysis on their functions. IMPORTANCE CAAX proteins are subject to multiple posttranslational modifications: cysteine prenylation, CAAX proteolysis, and carboxylmethylation. For investigations of CAAX proteolysis, this study took the novel approach of using a proteolysis-resistant CAAX sequence to stage Saccharomyces cerevisiae Ras2 GTPase in a farnesylated and nonproteolyzed state. Our approach specifically limited the effects of disrupting CAAX proteolysis to Ras2. This represented an improvement over previous methods where CAAX proteolysis was inhibited by gene knockout, small interfering RNA knockdown, or biochemical inhibition of the Rce1 CAAX protease, which can lead to pleiotropic and unclear attribution of effects due to the action of Rce1 on multiple CAAX proteins. Our approach yielded results that demonstrated specific impacts of CAAX proteolysis on the function, localization, and other properties of Ras2, highlighting the utility of this approach for investigating the impact of CAAX proteolysis in other protein contexts.
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DOI：
10.1073/pnas.92.7.2984
发表时间：
1995-03-28
影响因子：
11.1
作者：
BHATTACHARYA, S;CHEN, L;POWERS, S
通讯作者：
POWERS, S
影响因子：
6
作者：
Cazzanelli G;Pereira F;Alves S;Francisco R;Azevedo L;Dias Carvalho P;Almeida A;Côrte-Real M;Oliveira MJ;Lucas C;Sousa MJ;Preto A
通讯作者：
Preto A
DOI：
10.1038/nrm3255
发表时间：
2011-12-22
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
--
影响因子：
8.8
作者：
Christiano R;Nagaraj N;Fröhlich F;Walther TC
通讯作者：
Walther TC
影响因子：
4
作者：
Hobbs, G. Aaron;Der, Channing J.;Rossman, Kent L.
通讯作者：
Rossman, Kent L.