Proximity labeling with CB5LP in Arabidopsis seedlings using TurboID

ABSTRACT: Endoplasmic reticulum (ER)-localized cytochrome P450 enzymes rely on membrane-bound redox partners to establish the P450 monooxygenase system, facilitating electron transfer from pyridine dinucleotide cofactors NADPH and/or NADH. The eukaryotic ER membrane houses two electron transfer pathways: one mediated by NADPH-dependent cytochrome P450 oxidoreductase (CPR) and the other by NADH-dependent cytochrome b5 reductase (CBR) and cytochrome b5 (CB5). In some cases, CB5 can also deliver electrons for P450 enzymes. These systems support P450s and other oxidases across various biological processes. The Arabidopsis thaliana genome encodes five canonical CB5 genes—AtCB5A (At1g26340), AtCB5B (At2g32720), AtCB5C (At2g46650), AtCB5D (At5g48810), and AtCB5E (At5g53560)—all of which encode tail-anchored proteins with an N-terminal heme-binding (cyt-b5) domain and a C-terminal transmembrane (TM) domain. Additionally, Arabidopsis harbors a non-canonical CB5-like protein (AtCB5LP, At1g60660) that shares 36–46% sequence identity with canonical CB5s but exhibits an inverted topology, with its TM domain at the N-terminus and the cyt-b5 domain at the C-terminus. Despite this distinct structural arrangement, the biochemical and physiological roles of AtCB5LP remain largely unknown. Loss of CB5LP in Arabidopsis leads to embryonic defects and post-embryonic lethality. To explore its biological function, we employed proximity labeling using TurboID-tagged CB5LP, which identified 34 potential cytochrome P450 interactors, including CYP51A2. Broad metabolite profiling revealed significant alterations in the phytosterol composition of CB5LP knockout mutants, with elevated 14α-methyl-sterols and reduced sitosterol and stigmasterol levels, underscoring CB5LP’s pivotal role in sterol 14α-demethylation. In vitro assays using yeast microsomal extracts confirmed that CB5LP is essential for CYP51A2 activity, while a mutant variant lacking electron transfer capability failed to support CYP51 function. Collectively, these findings establish CB5LP as a critical electron donor required for CYP51-mediated phytosterol biosynthesis.

INSTRUMENT(S):

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Whole Body

SUBMITTER: Xianhai Zhao  

LAB HEAD: Chang-Jun Liu

PROVIDER: PXD060781 | Pride | 2025-09-10

REPOSITORIES: Pride