Protein Isoprenylation in Yeast Targets COOH-Terminal Sequences Not Adhering to the CaaX Consensus
Protein Isoprenylation in Yeast Targets COOH-Terminal Sequences Not Adhering to the CaaX Consensus
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DOI：
10.1534/genetics.118.301454
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发表时间：
2018-12-01
期刊：
影响因子：
3.3
通讯作者：
Schmidt, Walter K.
中科院分区：
文献类型：
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作者：
Berger, Brittany M.;Kim, June H.;Schmidt, Walter K.
蛋白质异戊二烯以COOH末端的Cxxx四肽序列的子集为靶点，该序列在操作上被定义为CAAX基序。然而，对于Cxxx序列的8000种可能组合中的每一种，法尼基转移酶的特异性在很大程度上仍未解决。在某种程度上，很难巩固来自体外和电子计算机方法的结果，这些方法产生的可预苯基化序列比体内已知的序列更广泛。我们已经研究了这种脱节是否是由于体内发生的对CAAX蛋白的翻译后修饰的多步骤复杂性造成的。例如，RAS GTP酶经历异戊二烯基化，然后在COOH末端发生额外的蛋白质分解和羧甲基化事件。相比之下，酿酒酵母Hsp40 Ydj1p是异戊二烯基化的，但不受额外修饰的影响。事实上，额外的修饰对Ydj1p在体内的活性是有害的。我们利用Ydj1p的性质和Ydj1p依赖的生长实验来鉴定允许Ydj1p在体内进行异戊二烯基化的序列，同时选择不能戊烯基化的和更广泛修改的序列。回收的序列与以前使用活体RAS酵母报告鉴定的序列基本不重叠。此外，现有的预测算法并不容易将大多数序列预测为异丙二烯基化靶标。我们的结果表明，酵母CAAX型戊烯基转移酶可以利用一系列超出传统CAAX蛋白限制的序列组合，这意味着可能比先前认为的更多的蛋白质被异戊二烯基化。
Protein isoprenylation targets a subset of COOH-terminal Cxxx tetrapeptide sequences that has been operationally defined as a CaaX motif. The specificity of the farnesyl transferase toward each of the possible 8000 combinations of Cxxx sequences, however, remains largely unresolved. In part, it has been difficult to consolidate results stemming from in vitro and in silico approaches that yield a wider array of prenylatable sequences relative to those known in vivo. We have investigated whether this disconnect results from the multistep complexity of post-translational modification that occurs in vivo to CaaX proteins. For example, the Ras GTPases undergo isoprenylation followed by additional proteolysis and carboxymethylation events at the COOH-terminus. By contrast, Saccharomyces cerevisiae Hsp40 Ydj1p is isoprenylated but not subject to additional modification. In fact, additional modifications are detrimental to Ydj1p activity in vivo. We have taken advantage of the properties of Ydj1p and a Ydj1p-dependent growth assay to identify sequences that permit Ydj1p isoprenylation in vivo while simultaneously selecting against nonprenylatable and more extensively modified sequences. The recovered sequences are largely nonoverlapping with those previously identified using an in vivo Ras-based yeast reporter. Moreover, most of the sequences are not readily predicted as isoprenylation targets by existing prediction algorithms. Our results reveal that the yeast CaaX-type prenyltransferases can utilize a range of sequence combinations that extend beyond the traditional constraints for CaaX proteins, which implies that more proteins may be isoprenylated than previously considered.