Yeast Split-Ubiquitin-Based Cytosolic Screening System to Detect Interactions Between Transcriptionally Active Proteins

Möckli, Natalie; Deplazes, Anna; Hassa, Paul O.; Zhang, Zhaolei; Peter, Matthias; Hottiger, Michael O.; Štagljar, Igor; Auerbach, Daniel

doi:10.2144/000112455

Cited by 74 publications

(74 citation statements)

References 20 publications

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“…To identify potential protein partners of full-length ZOU protein, a yeast two-hybrid screen was carried out using a split ubiquitinbased system in which the bait protein is presented as a membrane-anchored fusion (Möckli et al, 2007). Two independent cDNA clones encoding similar truncated versions of the bHLH protein INDUCER OF CBP EXPRESSION 1 (ICE1, also known as SCREAM; AT3g26744) were obtained in a screen of 1.2 million clones ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1

et al. 2014

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In Arabidopsis seeds, embryo growth is coordinated with endosperm breakdown. Mutants in the endosperm-specific gene ZHOUPI (ZOU), which encodes a unique basic helix-loop-helix (bHLH) transcription factor, have an abnormal endosperm that persists throughout seed development, significantly impeding embryo growth. Here we show that loss of function of the bHLH-encoding gene INDUCER OF CBP EXPRESSION 1 (ICE1) causes an identical endosperm persistence phenotype. We show that ZOU and ICE1 are co-expressed in the endosperm and interact in yeast via their bHLH domains. We show both genetically and in a heterologous plant system that, despite the fact that both ZOU and ICE1 can form homodimers in yeast, their role in endosperm breakdown requires their heterodimerization. Consistent with this conclusion, we confirm that ZOU and ICE1 regulate the expression of common target genes in the developing endosperm. Finally, we show that heterodimerization of ZOU and ICE1 is likely to be necessary for their binding to specific targets, rather than for their nuclear localization in the endosperm. By comparing our results with paradigms of bHLH function and evolution in animal systems we propose that the ZOU/ICE1 complex might have ancient origins, acquiring novel megagametophyte-specific functions in heterosporous land plants that were conserved in the angiosperm endosperm.

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Section: Resultsmentioning

confidence: 99%

“…Yeast two-hybrid experiments were carried out using the Dual Hunter split ubiquitin-based system from Dualsystems Biotech (Schlieren, Switzerland) (Möckli et al, 2007). All yeast protocols were performed exactly as described in the manufacturer's instructions.…”

Section: Yeast Two-hybrid Screenmentioning

confidence: 99%

Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1

et al. 2014

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“…One requirement of the SUS approach is that the bait protein be membrane anchored so that the bait construct cannot migrate to the nucleus on its own to activate the reporter gene in the absence of prey interaction. The CytoSUS assay overcomes this limitation, incorporating, as an N-terminal anchor for a soluble bait construct, the integral membrane protein subunit of the yeast oligosaccharyltransferase complex of the endoplasmic reticulum lumen (OST4p) (Möckli et al, 2007). We designed a Gateway-compatible SUS bait vector with this construct, here designated mOST4, to give expression with SEC11.…”

Section: Sec11 Rescues Secretory Block In Syp122 But Not In Syp121 Mumentioning

confidence: 99%

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane

et al. 2015

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SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.

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“…If bait and prey interact, Cub and NubG complement to form split-ubiquitin. Then, ubiquitin-specific proteases release LexA-VP16, which migrates to the nucleus and activates the transcription of reporter genes (Mö ckli et al, 2007). We screened an Arabidopsis seedling cDNA library for proteins that interact with PNM1.…”

Section: Pnm1 Interacts With Nuclear Proteinsmentioning

confidence: 99%

An Arabidopsis Dual-Localized Pentatricopeptide Repeat Protein Interacts with Nuclear Proteins Involved in Gene Expression Regulation

et al. 2011

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Following the endosymbiotic acquisition of mitochondria by eukaryotic cells, most of the genes in this organelle were transferred to the nucleus. To maintain mitochondrial biogenesis and function, nuclear and mitochondrial genomes require regulated and coordinated expression. In plant organelles, nuclear-encoded proteins targeted to the organelles control posttranscriptional and posttranslational mechanisms. Pentatricopeptide repeat (PPR) proteins are good candidates to play such regulatory roles. Here, we identify PNM1 (for PPR protein localized to the nucleus and mitochondria 1), a novel PPR protein that is dual localized to mitochondria and nuclei in Arabidopsis thaliana, as observed by green fluorescent protein fusions and immunodetection on subcellular fractions and on histological sections. Genetic complementation showed that loss of PNM1 function in mitochondria, but not in nuclei, is lethal for the embryo. In mitochondria, it is associated with polysomes and may play a role in translation. A genetic screen in yeast identified protein partners of PNM1. These partners, the nucleosome assembly protein NAP1, and the transcription factor TCP8 interact with PNM1 in the nucleus in planta. Furthermore, TCP8 can bind the promoter of PNM1. This suggests that PNM1 might be involved in the regulation of its own gene expression in the nucleus and could thus play a role in gene expression adjustments between mitochondria and the nucleus.

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Yeast Split-Ubiquitin-Based Cytosolic Screening System to Detect Interactions Between Transcriptionally Active Proteins

Cited by 74 publications

References 20 publications

Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1

Endosperm breakdown in Arabidopsis requires heterodimers of the basic helix-loop-helix proteins ZHOUPI and INDUCER OF CBP EXPRESSION 1

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane

An Arabidopsis Dual-Localized Pentatricopeptide Repeat Protein Interacts with Nuclear Proteins Involved in Gene Expression Regulation

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