Abstract

The enzyme Farnesyl pyrophosphate Synthase (FPS) plays a critical role in the production process of Eucommia ulmoides rubber (Eu-rubber), catalyzing the production of farnesyl pyrophosphate (FPP), the direct precursor for rubber formation. However, the ubiquitination modification of EuFPS, a key enzyme in this pathway, remains unexplored. Here, we identified ubiquitination-associated enzymes interacting with EuFPS1 and characterized its ubiquitination profile. Using protein interaction analysis, subcellular localization, in vivo and in vitro ubiquitination assays, and ELISA, our findings indicate that EuFPS1 is inherently unstable and undergoes degradation via the 26S proteasome. Its ubiquitination is regulated by the E2/E3 complex comprising EuUBC16, EuUev1a, and EuPUB21/24. Specifically, the EuUBC16-EuUev1a complex catalyzes K48- and K63-linked polyubiquitination of EuFPS1. Functional studies revealed that overexpression of EuUBC16 in Nicotiana benthamiana significantly increased both the content and activity of FPS, whereas MG132 treatment decreased them. This antagonistic effect suggests that the EuUBC16-EuUev1a complex regulates EuFPS1 stability and degradation. We provide the first insight into the ubiquitination regulation mechanism of the key enzyme EuFPS1 in Eu-rubber biosynthesis, offering a novel theoretical foundation and research direction for enhancing Eu-rubber content.