Arabidopsis Membrane-anchored Ubiquitin-fold (MUB) Proteins Localize a Specific Subset of Ubiquitin-conjugating (E2) Enzymes to the Plasma Membrane

Dowil, Rebecca T.; Lü, Xiaolong; Saracco, Scott A.; Vierstra, Richard D.; Downes, Brian P.

doi:10.1074/jbc.m110.158808

Cited by 29 publications

(27 citation statements)

References 57 publications

(76 reference statements)

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“…A specific subset of Arabidopsis UBC enzymes, subgroup VI, which includes UBC8 to 11 and UBC28 to 30, was found to be recruited to the PM through interaction with membraneanchored ubiquitin-fold proteins (Dowil et al, 2011). Despite our finding of membrane localization of PHO2, there is no transmembrane-helix domain or posttranslational modification sites predicted for membrane anchoring within PHO2.…”

Section: Pho2 Facilitates the Degradation Of Pht1 Proteins At The Emcontrasting

confidence: 50%

Identification of Downstream Components of Ubiquitin-Conjugating Enzyme PHOSPHATE2 by Quantitative Membrane Proteomics in Arabidopsis Roots

et al. 2013

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ORCID ID: 0000-0001-5953-4144 (T.-J.C.).MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/PHOSPHATE2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)-based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in PHOSPHATE TRANSPORTER1 (PHT1) family and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.

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Section: Pho2 Facilitates the Degradation Of Pht1 Proteins At The Emcontrasting

confidence: 50%

Identification of Downstream Components of Ubiquitin-Conjugating Enzyme PHOSPHATE2 by Quantitative Membrane Proteomics in Arabidopsis Roots

et al. 2013

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“…As an E3 ligase, FLY1 could mark PMTs with monoubiquitin for retention in the Golgi since monoubiquitin has been identified as a signal for membrane protein localization in yeast, animal, and plant systems (Reggiori and Pelham, 2002;Hicke and Dunn, 2003;Schnell andHicke, 2003, Barberon et al, 2011;Dowil et al, 2011). Alternatively, the FLY1 E3 ligase could use polyubiquitin chains to target PME proteins for degradation.…”

Section: Discussionmentioning

confidence: 99%

FLYING SAUCER1 Is a Transmembrane RING E3 Ubiquitin Ligase That Regulates the Degree of Pectin Methylesterification inArabidopsisSeed Mucilage

et al. 2013

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Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca 2+ ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca 2+ or completely rescued using alkaline Ca 2+ chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1-yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells.

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“…Additionally, components other than E3 ligases that interact with the these E2s also might contribute to the regulation of plant immunity. The membrane-anchored ubiquitin fold (MUB) proteins were found to interact specifically with the Arabidopsis counterparts of tomato group III E2 enzymes (Dowil et al, 2011). Considering the critical importance of group III E2s for PTI as shown here and that the PRRs that recognize MAMPs in PTI are localized to the plasma membrane, it would be intriguing to find out whether the MUB proteins cooperate with the group III E2 enzymes, particularly the E2s UBC11, UBC28, UBC29, UBC39, and UBC40, and their cognate E3 ubiquitin ligases to modify certain PRRs and, thus, play a role in regulating PTI.…”

Section: Discussionmentioning

confidence: 99%

A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity

et al. 2016

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Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart.Ubiquitination as a major posttranslational modification of proteins in eukaryotes has emerged in recent years as an important regulatory mechanism underlying plant innate immunity Fu et al., 2012;Marino et al., 2012;Li et al., 2014). The ubiquitination process involves a consecutive, threestep enzymatic cascade that is catalyzed by three different classes of enzymes: ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin ligase (E3) enzymes. The first step of the process activates ubiquitin, a highly conserved 76-amino acid protein, in an ATP-dependent manner by attaching ubiquitin to an E1 enzyme. The activated ubiquitin is then transferred from the E1 to the Cys residue at the active site of an E2 conjugating enzyme. An E3 ligase then recruits the substrate protein to the E2 to transfer the ubiquitin molecule from E2 to the substrate. Through the action of E1, E2, and E3, ubiquitin is covalently attached usually to the Lys residue of a substrate through an isopeptide bond (Hershko and Ciechanover, 1998). Of the three enzymes involved in ubiquitination, E3 ubiquitin ligases have been the focus of many studies due to their key role in determining substrate specificity for the ubiquitination

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Arabidopsis Membrane-anchored Ubiquitin-fold (MUB) Proteins Localize a Specific Subset of Ubiquitin-conjugating (E2) Enzymes to the Plasma Membrane

Cited by 29 publications

References 57 publications

Identification of Downstream Components of Ubiquitin-Conjugating Enzyme PHOSPHATE2 by Quantitative Membrane Proteomics in Arabidopsis Roots

Identification of Downstream Components of Ubiquitin-Conjugating Enzyme PHOSPHATE2 by Quantitative Membrane Proteomics in Arabidopsis Roots

FLYING SAUCER1 Is a Transmembrane RING E3 Ubiquitin Ligase That Regulates the Degree of Pectin Methylesterification inArabidopsisSeed Mucilage

A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity

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