Investigation of Membrane Protein—protein Interactions Using Correlative FRET-PLA

Ivanusic, Daniel; Eschricht, Magdalena; Denner, Joachim

doi:10.2144/000114215

Cited by 22 publications

(39 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We next employed an in situ proximity ligation assay (PLA) in which separate probes are used for two proteins within a cell and a fluorescent signal is obtained only if the two proteins are in close enough proximity for direct protein-protein interactions (< 40 nm)(Ivanusic et al, 2014; Jarvius et al, 2007; Soderberg et al, 2006). The in situ PLA produced compelling results showing WBSCR16 and OPA1 to be in extremely close proximity in situ , within the cell (Fig.…”

Section: Resultsmentioning

confidence: 99%

WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion

et al. 2017

View full text Add to dashboard Cite

SUMMARY Regulated inter-mitochondrial fusion/fission is essential for maintaining optimal mitochondrial respiration and control of apoptosis and autophagy. In mammals, mitochondrial fusion is controlled by outer membrane GTPases MFN1 and MFN2, and by inner membrane GTPase OPA1. Disordered mitochondrial fusion/fission contributes to various pathologies, and MFN2 or OPA1 mutations underlie neurodegenerative diseases. Here, we show that the WBSCR16 protein is primarily associated with the outer face of the inner mitochondrial membrane and is important for mitochondrial fusion. We provide evidence of a WBSCR16/OPA1 physical interaction in the intact cell and of a WBSCR16 function as an OPA1-specific guanine nucleotide exchange factor (GEF). Homozygosity for a Wbscr16 mutation causes early embryonic lethality, whereas neurons of mice heterozygous for the mutation have mitochondria with reduced membrane potential and increased susceptibility to fragmentation upon exposure to stress, suggesting roles for WBSCR16 deficits in neuronal pathologies.

show abstract

Section: Resultsmentioning

confidence: 99%

WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion

et al. 2017

View full text Add to dashboard Cite

show abstract

“…The principle of this technique is that energy transfer is eliminated (or reduced) when the acceptor is bleached, thus yielding an increase in donor fluorescence, which is a measure of FRET efficiency. To detect FRET, we monitored the donor and acceptor fluorescence intensities before, during, and after the acceptor photobleaching procedure (36). We used either RFP-fusion protein or a primary antibody followed by tetramethylrhodamine (TRITC)-conjugated secondary antibody as acceptor, whereas GFP-fusion protein or primary antibody/FITC-conjugated secondary antibody was used as donor.…”

Section: Fluorescence Resonance Energy Transfer Reveals the Proximitymentioning

confidence: 99%

VAMP-associated protein-A and oxysterol-binding protein–related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum

Santos

Rappa

Karbanová

et al. 2018

Journal of Biological Chemistry

View full text Add to dashboard Cite

The endocytic pathway plays an instrumental role in recycling internalized molecules back to the plasma membrane or in directing them to lysosomes for degradation. We recently reported a new role of endosomes-the delivery of components from extracellular vesicles (EVs) to the nucleoplasm of recipient cells. Using indirect immunofluorescence, FRET, immunoisolation techniques, and RNAi, we report here a tripartite protein complex (referred to as the VOR complex) that is essential for the nuclear transfer of EV-derived components by orchestrating the specific localization of late endosomes into nucleoplasmic reticulum. We found that the VOR complex contains the endoplasmic reticulum-localized vesicle-associated membrane protein (VAMP)-associated protein A (VAP-A), the cytoplasmic oxysterol-binding protein-related protein 3 (ORP3), and late endosome-associated small GTPase Rab7. The silencing of VAP-A or ORP3 abrogated the association of Rab7-positive late endosomes with nuclear envelope invaginations and, hence, the transport of endocytosed EV-derived components to the nucleoplasm of recipient cells. We conclude that the VOR complex can be targeted to inhibit EV-mediated intercellular communication, which can have therapeutic potential for managing cancer in which the release of EVs is dysregulated.

show abstract

“…CD63 is a tetraspanin that contains four transmembrane domains (a 4TM protein). This protein is predominantly localized in the plasma membrane (22, 23), and we identified it as an interacting partner of gp41 (24). In addition to CD63, another 4TM protein, the emopamil binding protein EBP (sterol isomerase), was also identified in earlier split-ubiquitin screens as a frequent interaction partner of gp41.…”

Section: Resultsmentioning

confidence: 97%

Improved Split-Ubiquitin Screening Technique to Identify Surface Membrane Protein-Protein Interactions

et al. 2015

Self Cite

View full text Add to dashboard Cite

Yeast-based methods are still the workhorse for the detection of protein-protein interactions (PPIs) in vivo. Yeast two-hybrid (Y2H) systems, however, are limited to screening for a specific group of molecules that interact in a particular cell compartment. For this reason, the split-ubiquitin system (SUS) was developed to allow screening of cDNA libraries of full-length membrane proteins for protein-protein interactions in Saccharomyces cerevisiae. Here we demonstrate that a modification of the widely used membrane SUS involving the transmembrane (TM) domain of the yeast receptor Wsc1 increases the stringency of screening and improves the selectivity for proteins localized in the plasma membrane (PM).

show abstract

Investigation of Membrane Protein—protein Interactions Using Correlative FRET-PLA

Cited by 22 publications

References 30 publications

WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion

WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion

VAMP-associated protein-A and oxysterol-binding protein–related protein 3 promote the entry of late endosomes into the nucleoplasmic reticulum

Improved Split-Ubiquitin Screening Technique to Identify Surface Membrane Protein-Protein Interactions

Contact Info

Product

Resources

About