A comprehensive in vivo screen of yeast farnesyltransferase activity reveals broad reactivity across a majority of CXXX sequences.
A comprehensive in vivo screen of yeast farnesyltransferase activity reveals broad reactivity across a majority of CXXX sequences.
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DOI：
10.1093/g3journal/jkad094
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发表时间：
2023-07-05
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目前对法尼基转移酶（FTase）特异性的理解是通过研究报告基因（如Ras和Ras相关蛋白）而开创的，这些报告基因具有由4个氨基酸残基组成的C-末端CaaX基序：半胱氨酸-组氨酸1-组氨酸2-可变（X）。这些研究导致发现具有CaaX基序的蛋白质经历3步翻译后修饰途径，包括法尼基化、蛋白水解和羧基甲基化。然而，新出现的证据表明，FTase可以法尼基化CaaX基序之外的序列，并且这些序列不经历典型的3步途径。在这项工作中，我们报告了一个全面的评估所有可能的CXXX序列作为FTase的目标，使用报告Ydj 1，热休克蛋白40分子伴侣，只需要法尼基化的活动。我们的遗传和高通量测序方法揭示了酵母FTase可以在体内识别的前所未有的序列概况，这有效地扩展了FTase在酵母蛋白质组中的潜在靶空间。我们还记录了酵母FTase特异性主要受α 2和X位置处的限制性氨基酸的影响，而不是如先前所认为的CaaX基序的相似性。对CXXX空间的首次完整评估扩展了蛋白质异戊二烯化的复杂性，并标志着在了解该异戊二烯化途径的潜在靶点范围方面向前迈出了关键一步。
The current understanding of farnesyltransferase (FTase) specificity was pioneered through investigations of reporters like Ras and Ras-related proteins that possess a C-terminal CaaX motif that consists of 4 amino acid residues: cysteine–aliphatic1–aliphatic2–variable (X). These studies led to the finding that proteins with the CaaX motif are subject to a 3-step post-translational modification pathway involving farnesylation, proteolysis, and carboxylmethylation. Emerging evidence indicates, however, that FTase can farnesylate sequences outside the CaaX motif and that these sequences do not undergo the canonical 3-step pathway. In this work, we report a comprehensive evaluation of all possible CXXX sequences as FTase targets using the reporter Ydj1, an Hsp40 chaperone that only requires farnesylation for its activity. Our genetic and high-throughput sequencing approach reveals an unprecedented profile of sequences that yeast FTase can recognize in vivo, which effectively expands the potential target space of FTase within the yeast proteome. We also document that yeast FTase specificity is majorly influenced by restrictive amino acids at a2 and X positions as opposed to the resemblance of CaaX motif as previously regarded. This first complete evaluation of CXXX space expands the complexity of protein isoprenylation and marks a key step forward in understanding the potential scope of targets for this isoprenylation pathway.
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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
影响因子：
4
作者：
Hobbs, G. Aaron;Der, Channing J.;Rossman, Kent L.
通讯作者：
Rossman, Kent L.
影响因子：
5.8
作者：
Firth, AE;Patrick, WM
通讯作者：
Patrick, WM
影响因子：
2.9
作者：
Hartman, HL;Hicks, KA;Fierke, CA
通讯作者：
Fierke, CA
影响因子：
3.3
作者：
Berger, Brittany M.;Kim, June H.;Schmidt, Walter K.
通讯作者：
Schmidt, Walter K.