A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins
            <i>in vivo</i>

Štagljar, Igor; Korostensky, Chantal; Johnsson, Nils; Heesen, Stephan te

doi:10.1073/pnas.95.9.5187

Cited by 560 publications

(473 citation statements)

References 23 publications

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“…To determine the actual membrane topology of human Tmem65, we employed a membrane yeast two-hybrid (MYTH) system, as previously described (Fig. 4c,d) [26][27][28] . To determine protein topography, both N-terminal (TF-Cub-Tmem65) and C-terminal (Tmem65-Cub-TF) 'bait' Tmem65 proteins were tested for protein-protein interactions (PPIs) in the presence of a non-interacting yeast integral membrane protein Ost1p (ref.…”

Section: Resultsmentioning

confidence: 99%

“…27) fused to either NubI (Ost1-NubI) or NubG (Ost1-NubG). The Ost1-NubI control 'prey' construct is designed to activate the yeast reporter system, irrespective of a PPI because of the high affinity with which NubI associated with Cub to form active ubiquitin 26 , but only if the Cub domain of the corresponding 'bait' protein is localized in the cytosol. Activation of the reporter system was measured as growth on media lacking histidine and adenine in the presence of X-gal.…”

Section: Methodsmentioning

confidence: 99%

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Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

et al. 2015

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Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts. Functional analysis of Tmem65 both in vitro using lentiviral shRNA-mediated knockdown in mouse cardiomyocytes and in vivo using morpholino-based knockdown in zebrafish show marked alterations in gap junction function and cardiac morphology. Molecular analyses suggest that Tmem65 interaction with connexin 43 (Cx43) is required for correct localization of Cx43 to the intercalated disc, since Tmem65 deletion results in marked internalization of Cx43, a shorter half-life through increased degradation, and loss of Cx43 function. Our data demonstrate that the membrane protein Tmem65 is an intercalated disc protein that interacts with and functionally regulates ventricular Cx43.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

et al. 2015

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“…Split ubiquitin-based yeast two-hybrid assays were performed using the yeast strain NMY32 supplied by Dualsystems Biotech 54 . The pCCW vector encoding the Cub-LexA-VP16 fragment was used to construct the bait plasmids.…”

Section: Methodsmentioning

confidence: 99%

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway

Ouyang

et al. 2011

Nat Commun

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Higher plants require chloroplasts for essential functions in photosynthesis and other important physiological processes, such as sugar, lipid and amino-acid biosynthesis. most chloroplast proteins are nuclear-encoded proteins that are synthesized in the cytosol as precursors, and imported into chloroplasts by protein translocases in the outer and inner chloroplast envelope. The imported chloroplast proteins are then translocated into or across the thylakoid membrane by four distinct pathways. However, the mechanisms by which the imported nuclear-encoded proteins are delivered to these pathways remain largely unknown. Here we show that an Arabidopsis ankyrin protein, LTD (mutation of which causes the light-harvesting chlorophyllbinding protein translocation defect), is localized in the chloroplast and using yeast two-hybrid screens demonstrate that LTD interacts with both proteins from the signal recognition particle (sRP) pathway and the inner chloroplast envelope. our study shows that LTD is essential for the import of light-harvesting chlorophyll-binding proteins and subsequent routing of these proteins to the chloroplast sRP-dependent pathway.

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“…The putative SUMO1-binding cDNAs were cloned into pMET-Ste14-C ub -RUra3, replacing yeast Ste14. AtSUMO1, AtSUMO1ΔGG, and AtSUMO3 cDNAs were cloned into modified versions of the pCup-N ub -Sec62 vector, replacing yeast Sec62, respectively (Stagljar et al, 1998). Interactions between each pair of proteins were tested on selective medium containing 2 mg/ml 5-FOA and selective medium lacking uracil.…”

Section: Yeast Split Ubiquitin Assaymentioning

confidence: 99%

Identification and Molecular Properties of SUMO-Binding Proteins in Arabidopsis

Park

Choi

Park

et al. 2011

Molecules and Cells

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Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes.

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A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo

Abstract: A detection system for interactions between membrane proteins in vivo is described. The system is based on split-ubiquitin

Cited by 560 publications

References 23 publications

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway

Identification and Molecular Properties of SUMO-Binding Proteins in Arabidopsis

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