Liposome-Binding Assays to Assess Specificity and Affinity of Phospholipid–Protein Interactions

Julkowska, Magdalena; Rankenberg, Johanna; Testerink, Christa

doi:10.1007/978-1-62703-401-2_24

Cited by 53 publications

(40 citation statements)

References 11 publications

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“…To test whether SnRK2.10 and its most related family member, SnRK2.4, directly bind to PA and to assess the specificity of lipid binding, recombinant GST–SnRK2.10 and GST–SnRK2.4 proteins purified from Escherichia coli were tested in a liposome binding assay (Figure 7b). Since liposomes consist of a lipid bilayer, interaction with a more naturally organized PA can be shown using this approach (Julkowska et al. , 2012; Testerink et al.…”

Section: Resultsmentioning

confidence: 99%

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The Snf1‐related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress

et al. 2012

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The sucrose non-fermenting-1-related protein kinase 2 (SnRK2) family represents a unique family of plant-specific protein kinases implicated in cellular signalling in response to osmotic stress. In our studies, we observed that two class 1 SnRK2 kinases, SnRK2.4 and SnRK2.10, are rapidly and transiently activated in Arabidopsis roots after exposure to salt. Under saline conditions, snrk2.4 knockout mutants had a reduced primary root length, while snrk2.10 mutants exhibited a reduction in the number of lateral roots. The reduced lateral root density was found to be a combinatory effect of a decrease in the number of lateral root primordia and an increase in the number of arrested lateral root primordia. The phenotypes were in agreement with the observed expression patterns of genomic yellow fluorescent protein (YFP) fusions of SnRK2.10 and -2.4, under control of their native promoter sequences. SnRK2.10 was found to be expressed in the vascular tissue at the base of a developing lateral root, whereas SnRK2.4 was expressed throughout the root, with higher expression in the vascular system. Salt stress triggered a rapid re-localization of SnRK2.4–YFP from the cytosol to punctate structures in root epidermal cells. Differential centrifugation experiments of isolated Arabidopsis root proteins confirmed recruitment of endogenous SnRK2.4/2.10 to membranes upon exposure to salt, supporting their observed binding affinity for the phospholipid phosphatidic acid. Together, our results reveal a role for SnRK2.4 and -2.10 in root growth and architecture in saline conditions.

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Section: Resultsmentioning

confidence: 99%

“…Bound protein was eluted from the glutathione agarose resin using elution buffer containing 20 m m reduced glutathione in 50 m m TRIS‐HCl pH 8.0. Liposome‐binding assays were performed as described in Julkowska et al. (2012).…”

Section: Methodsmentioning

confidence: 99%

The Snf1‐related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress

et al. 2012

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“…For instance, numerous proteins have been proposed to contain PIbinding domains, making these proteins prime candidates for regulation by PIs (van Leeuwen et al, 2004). However, only a minority of these potential PI-modulins have been experimentally tested for PI binding by in vitro lipid overlay assays (Munnik and Wierzchowiecka, 2013) or liposome binding tests (Julkowska et al, 2013), leaving the field open for further scrutiny. Whereas many of the potential PI-modulins are proteins of unknown function, others are well annotated, linking potential PI binding to relevant cellular processes.…”

Section: Pi Functions In Plantsmentioning

confidence: 99%

Phosphoinositide signaling in plant development

2016

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The membranes of eukaryotic cells create hydrophobic barriers that control substance and information exchange between the inside and outside of cells and between cellular compartments. Besides their roles as membrane building blocks, some membrane lipids, such as phosphoinositides (PIs), also exert regulatory effects. Indeed, emerging evidence indicates that PIs play crucial roles in controlling polarity and growth in plants. Here, I highlight the key roles of PIs as important regulatory membrane lipids in plant development and function.

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“…Liposome binding assays were performed as previously described (Julkowska et al, 2013). Liposomes were prepared from 400 nmol of total lipids at the following molar ratios: PtdCho:PtdEtn, 1:1; PtdCho: PtdEtn:PtdIns4P, 2:2:1; PtdCho:PtdEtn:PtdIns(4,5)P 2 , 2:2:1; PtdCho: PtdEtn:PtdOH, 2:2:1.…”

Section: Phospholipid Binding Assaysmentioning

confidence: 99%

Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses

et al. 2016

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Polar transport of the phytohormone auxin through PIN-FORMED (PIN) auxin efflux carriers is essential for the spatiotemporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it colocalizes with PINs and activates PIN-mediated auxin efflux. Here, we show that the association of D6PK with the basal plasma membrane and PINs is dependent on the phospholipid composition of the plasma membrane as well as on the phosphatidylinositol phosphate 5-kinases PIP5K1 and PIP5K2 in epidermis cells of the primary root. We further show that D6PK directly binds polyacidic phospholipids through a polybasic lysine-rich motif in the middle domain of the kinase. The lysine-rich motif is required for proper PIN3 phosphorylation and for auxin transport-dependent tropic growth. Polybasic motifs are also present at a conserved position in other D6PK-related kinases and required for membrane and phospholipid binding. Thus, phospholipid-dependent recruitment to membranes through polybasic motifs might not only be required for D6PK-mediated auxin transport but also other processes regulated by these, as yet, functionally uncharacterized kinases.

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Liposome-Binding Assays to Assess Specificity and Affinity of Phospholipid–Protein Interactions

Cited by 53 publications

References 11 publications

The Snf1‐related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress

The Snf1‐related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress

Phosphoinositide signaling in plant development

Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses

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