| 摘要: | 蛋白質異戊二烯化（protein prenylation），包括法尼脂化（farnesylation）與香葉基化（geranylgeranylation），是一類重要的蛋白質脂質修飾機制。含有 CaaX 端序列的蛋白質會由異二聚酶組成的法尼基轉移酶（PFT）或香葉基轉移酶
（PGGT-I）進行修飾，而此類修飾在植物中對蛋白質細胞內的定位、穩定性與功能，尤其在逆境反應中，具有關鍵調控作用。在 Arabidopsis thaliana 中，HSP40 蛋白 AtJ3 需要接受法尼脂化，才可以與HSP70-4於stress granule協同運作並提供耐熱性；然而，香葉基化是否能在熱耐受性方面取代法尼脂化，仍未知曉。
在本研究中，我們於 Escherichia coli 中共表現 AtPGGTα、AtPGGTβ 以及 AtGGPPS2，提供功能性的香葉基焦磷酸（GGPP）以建立植物蛋白質香葉基化系統。利用此系統，我們確認三種CaaX修改過的 AtJ3（AtJ3CHLF、AtJ3CSFL、AtJ3CAFL）能被AtPGGT辨識並進行修飾。此外，透過先前研究於E. coli建立的植物法尼脂化辨識系統，我們也發現 AtJ3CHLF 與 AtJ3CSFL可在 E. coli 中接受法尼脂化，顯示其具有被法尼脂化也具有被香葉基化修飾的能力，然而AtJ3CAFL則只具有被香葉基化修飾的能力。
將這些修改的 AtJ3 的基因導入 Arabidopsis j3 突變株後所得之轉殖株，在耐熱試驗中皆顯示三種轉殖株熱耐受性皆弱於野生型 AtJ3，顯示香葉基化在維持 AtJ3 所介導的耐熱功能上效果較差。也因此影響了轉殖株的耐熱能力。
綜合而言，本研究揭示 CaaX 序列組成會決定 AtJ3 所接受的異戊二烯修飾類型，並深刻影響其熱耐受性。我們建立的細菌系統提供一個研究植物蛋白質香葉基化的新平台，同時也提出明確證據指出：相較於香葉基化，法尼脂化對植物獲得最佳熱耐受性更為關鍵。
;Protein prenylation, including farnesylation and geranylgeranylation. Protein with N-terminal CaaX motif is catalyzed by a heterodimeric protein farnesyltransferase (PFT), geranylgeranyltransferase (PGGT-I). It is a crucial post-translational lipid modification that regulates protein localization, stability, and function in plant stress responses. In Arabidopsis thaliana, the HSP40 chaperone AtJ3 requires farnesylation to cooperate with HSP70-4 within stress granules and thereby confer thermotolerance. However, whether geranylgeranylation can functionally substitute for farnesylation for heat tolerance remains unknown.
In this study, we co-expressed AtPGGTα, AtPGGTβ, and AtGGPPS2 in Escherichia coli to generate a functional supply of geranylgeranyl pyrophosphate (GGPP) and thereby establish a plant protein geranylgeranylation system. Using this platform, we demonstrated that three CaaX-modified AtJ3 variants (AtJ3CHLF, AtJ3CSFL, and AtJ3CAFL) were recognized and modified by AtPGGT. In addition, by employing a previously established plant protein farnesylation system in E. coli, we found that AtJ3CHLF and AtJ3CSFL, but not AtJ3CAFL, could also undergo farnesylation, indicating alternative prenylation potential for AtJ3CHLF and AtJ3CSFL, whereas AtJ3CAFL is restricted to geranylgeranylation. Transgenic Arabidopsis j3 mutant lines expressing full-genomic engineered AtJ3 (AtJ3CHLF, AtJ3CSFL, and AtJ3CAFL) exhibited reduced thermotolerance compared with wild-type AtJ3, suggesting that geranylgeranylation is less effective than farnesylation in maintaining AtJ3-mediated heat protection. Additional assays revealed that AtJ3CHLF and AtJ3CSFL, but not AtJ3CAFL, could also be farnesylated in E. coli, indicating AtJ3CHLF and AtJ3CSFL have ability of both farnesylation and geranylgeranylation, AtJ3CAFL have ability of geranylgeranylation. These findings demonstrate that CaaX motif composition critically determines the type of prenylation and its functional consequences. This study establishes a bacterial platform for analyzing plant protein geranylgeranylation and provides mechanistic evidence that farnesylation, rather than geranylgeranylation, is essential for optimal thermotolerance in plants. |
