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. 2021 Oct 19;10(10):2228.
doi: 10.3390/plants10102228.

Unique N-Terminal Interactions Connect F-BOX STRESS INDUCED (FBS) Proteins to a WD40 Repeat-like Protein Pathway in Arabidopsis

Edgar Sepulveda-Garcia  1   2 , Elena C Fulton  3 , Emily V Parlan  3 , Lily E O'Connor  3 , Anneke A Fleming  3 , Amy J Replogle  3 , Mario Rocha-Sosa  2 , Joshua M Gendron  4 , Bryan Thines  3
Affiliations

Unique N-Terminal Interactions Connect F-BOX STRESS INDUCED (FBS) Proteins to a WD40 Repeat-like Protein Pathway in Arabidopsis

Edgar Sepulveda-Garcia et al. Plants (Basel). .

Abstract

SCF-type E3 ubiquitin ligases provide specificity to numerous selective protein degradation events in plants, including those that enable survival under environmental stress. SCF complexes use F-box (FBX) proteins as interchangeable substrate adaptors to recruit protein targets for ubiquitylation. FBX proteins almost universally have structure with two domains: A conserved N-terminal F-box domain interacts with a SKP protein and connects the FBX protein to the core SCF complex, while a C-terminal domain interacts with the protein target and facilitates recruitment. The F-BOX STRESS INDUCED (FBS) subfamily of plant FBX proteins has an atypical structure, however, with a centrally located F-box domain and additional conserved regions at both the N- and C-termini. FBS proteins have been linked to environmental stress networks, but no ubiquitylation target(s) or biological function has been established for this subfamily. We have identified two WD40 repeat-like proteins in Arabidopsis that are highly conserved in plants and interact with FBS proteins, which we have named FBS INTERACTING PROTEINs (FBIPs). FBIPs interact exclusively with the N-terminus of FBS proteins, and this interaction occurs in the nucleus. FBS1 destabilizes FBIP1, consistent with FBIPs being ubiquitylation targets SCFFBS1 complexes. This work indicates that FBS proteins may function in stress-responsive nuclear events, and it identifies two WD40 repeat-like proteins as new tools with which to probe how an atypical SCF complex, SCFFBS, functions via FBX protein N-terminal interaction events.

Keywords: F-box protein; SCF complex; WD40 repeat-like protein; stress response.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
The Arabidopsis F-BOX STRESS INDUCED (FBS) protein family. (A) Full-length protein sequence alignment of the four Arabidopsis FBS family members (FBS1–FBS4) created with the T-COFFEE sequence alignment program. Asterisks are fully conserved residues, colons are strongly conserved residue properties, and periods are weakly conserved residue properties. (B) FBS family interactions with ASK1 in yeast two-hybrid assays. Diploid yeast strains with indicated test constructs as bait (DBD) and prey (AD) were grown in liquid culture, diluted (OD600 = 100, 10−1, 10−2, 10−3), and spotted on SD medium minus Trp/Leu (-TL), minus Trp/Leu/His (-TLH), and minus Trp/Leu/His/Ade (-TLHA).
Figure 2
Figure 2
FBS INTERACTING PROTEIN (FBIP) sequence features. Full-length protein sequence alignment of the two Arabidopsis FBIP family members created with the T-COFFEE sequence alignment program. Blue indicates locations of seven WD40-like repeat sequences predicted by the WD40-repeat protein Structure Predictor version 2.0 (WDSPdb 2.0). Asterisks are fully conserved residues, colons are strongly conserved residue properties, and periods are weakly conserved residue properties.
Figure 3
Figure 3
Yeast two-hybrid (Y2H) interactions between FBS1 and FBIP proteins. (A) Full-length FBS1 interactions with full-length FBIP1 and FBIP2. Diploid yeast strains with indicated test constructs as bait (DBD) and prey (AD) were grown in liquid culture, diluted (OD600 = 100, 10−1, 10−2, 10−3), and spotted on SD medium minus Trp/Leu (-TL), minus Trp/Leu/His (-TLH), and minus Trp/Leu/His/Ade (-TLHA). (B) Truncated FBS1 bait (DBD) construct interaction with full length FBIP1 prey (AD). Amino acid deletions are indicated on the left.
Figure 4
Figure 4
Bimolecular fluorescence complementation (BiFC) interactions between FBS1 and FBIP proteins. Laser-scanning confocal microscopy of N. benthamiana epidermal cells expressing N-terminal nYFP- or cYFP-tagged FBS1 and FBIP proteins. FBS1 interactions with FBIP1 (top row) or FBIP2 (bottom row) are visualized on the BiFC yellow channel (YFP, left column). A co-expressed H2B-RFP (as nuclear marker) is visualized on the red channel (RFP, middle column) and YFP/RFP images are overlaid (Merge, right column). Arrow indicates selected nuclei in the expanded inset image. Scale bar = 100 µm.
Figure 5
Figure 5
FBS1 influence on FBIP1 protein abundance in plants. N. benthamiana leaves were infiltrated with Agrobacterium (C58C1) strains to express the tagged proteins. Agrobacterium mixes contained varying cell densities of strains harboring expression constructs (myc-FBS1 and/or FBIP1-HA), a suppressor protein (p19) or untransformed cells. Total protein was isolated from leaves 3 days after infiltration, separated by SDS-PAGE, transferred, and probed with antibodies against myc (top row, FBS1) or HA (second row, FBIP1). Bottom two rows show Ponceau S staining of the major subunit of Rubisco from the same two blots as a loading control.
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