Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis

Shyu, Y. John; Hiatt, Susan M.; Duren, Holli M.; Ellis, Ronald; Kerppola, Tom K.; Hu, Chang

doi:10.1038/nprot.2008.16

Cited by 78 publications

(80 citation statements)

References 31 publications

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“…To test this hypothesis, we used BiFC analysis to look at association between SUP-1 and UNC-17 in the C. elegans cholinergic nervous system. This approach has been used in a number of organisms, including C. elegans, to visualize protein-protein interactions (Hu et al 2002;Shyu et al 2008). For these experiments, an N-terminal partial peptide from YFP (NYFP) was fused to the N terminus of UNC-17, and a C-terminal partial peptide from CFP (CCFP) was fused to the C terminus of SUP-1.…”

Section: Effects Of Sup-1 Mutations On Unc-17 Abundance and Traffickingmentioning

confidence: 99%

“…We also show that the G347R mutation in TMD9 of UNC-17 does not prevent close association with the wild-type SUP-1 protein at synapses ( Figure 7C). BiFC data are normally interpreted as a measure of specific protein-protein interactions (Hu et al 2002;Shyu et al 2008). Alternatively, the observed BiFC interactions may actually reflect protein proximity and not necessarily protein interaction (Bhat et al 2006).…”

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confidence: 99%

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Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

et al. 2012

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The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-offunction mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-toarginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/ VAChT and synaptobrevin. C OORDINATED muscle contraction in both vertebrates and nematodes requires release of the neurotransmitter acetylcholine (ACh) from motor neurons. ACh is synthesized in motor neurons by the enzyme choline acetyltransferase (ChAT) and transported into synaptic vesicles by the vesicular ACh transporter (VAChT). Following transmitter release, cholinergic signaling is terminated primarily through the action of acetylcholinesterase (AChE), which hydrolyses the released ACh into choline and acetyl-CoA. In the nematode Caenorhabditis elegans, the ChAT and VAChT proteins are encoded by the cha-1 and unc-17 genes, respectively (Alfonso et al. , 1994b. Mutations that functionally eliminate either protein are lethal , while reduction-of-function mutations result in animals that are small, slow growing, uncoordinated, and resistant to AChE inhibitors such as aldicarb .The unc-17 missense mutation e245 replaces a glycine with an arginine (G347R) in the ninth transmembrane domain (TMD9) of the VAChT protein . Genetic screens designed to identify mutations that improve the locomotion of e245 homozygotes have identified several loci that suppress the deleterious effects of the VAChT G347R mutation. Sandoval et al. (2006) found that the sup-8 locus corresponds to the previously characterized snb-1 gene, which encodes the v-SNARE synaptobrevin. The single sup-8 allele is associated with a missense mutation that results in a charge alteration (I97D) in the TMD of synaptobrevin. T...

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Section: Effects Of Sup-1 Mutations On Unc-17 Abundance and Traffickingmentioning

confidence: 99%

mentioning

confidence: 99%

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

et al. 2012

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“…To investigate the cellular interactions between USP8 and the ESCRT-0 complex, BiFC microscopy was employed. BiFC takes advantage of a GFP variant, Venus fluorescent protein (VFP) to enable visualization of protein-protein complexes as they form in living cells (35,38,39). In this assay, the VFP fluorophore is separated into N-and C-terminal fragments (VN and VC, respectively), each fused to one of two proteins of interest (Fig.…”

Section: Usp8 Modulates the Integrity Of The Endocytic Compartment Atmentioning

confidence: 99%

The Deubiquitinating Enzyme USP8 Promotes Trafficking and Degradation of the Chemokine Receptor 4 at the Sorting Endosome

Berlin¹,

Higginbotham

Dise

et al. 2010

Journal of Biological Chemistry

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Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the earlyto-late endosome transition.Endocytosis and trafficking of cell surface receptors is essential for organized signal transduction and maintenance of homeostasis. Malfunctions along the molecular pathways governing endocytosis can lead to a wide range of human pathologies including metabolic disorders, autoimmune diseases, and cancer (1, 2). Ubiquitination, a reversible post-translational modification of proteins (3), mediates spatial and temporal aspects of endocytosis by dictating macromolecular complex assembly and cargo fate (4). Although polyubiquitination linked through Lys 48 of ubiquitin targets substrates for proteasomal degradation (5), mono-and Lys 63 -linked ubiquitination signal cargo trafficking (6), and modulate function of endocytic sorting machinery (7). The reversible nature of ubiquitination is imparted by deubiquitinating enzymes (DUBs) 4 and enables dynamic regulation of these ubiquitin-dependent processes in the cell (8).Trafficking of endocytosed cargo for degradation by the lysosome occurs in a series of discrete selection stages that utilize the endosomal sorting complexes required for transport (ESCRTs) -0, -I, -II, and -III. The ESCRT-0 complex, consisting of the adaptor proteins hepatocyte growth factor-regulated substrate (Hrs) and signal transducing adaptor molecule (STAM), resides at the sorting endosome and functions in the first step of cargo selection. ESCRT-0 partitions the endocytosed material arriving on the early endosomes between recycling and the multivesicular body (9 -11) and interacts with downstream ESCRT machinery (12, 13) respon...

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“…Like mammalian and Drosophila Fos and Jun proteins, C. elegans FOS-1 and JUN-1 form heterodimers Shyu et al, 2008). To see whether C. elegans jun-1 has the same role as fos-1, we performed RNAi in the wild type and failed to observe any phenotype in the F1 progeny, possibly because jun-1 mRNA was not eliminated (unpublished observations).…”

Section: Knockdown Of Fos-1 and Jun-1 Causes Sterilitymentioning

confidence: 99%

Caenorhabditis elegans FOS-1 and JUN-1 Regulateplc-1Expression in the Spermatheca to Control Ovulation

Hiatt

Duren

Shyu

et al. 2009

MBoC

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Fos and Jun are components of activator protein-1 (AP-1) and play crucial roles in the regulation of many cellular, developmental, and physiological processes. Caenorhabditis elegans fos-1 has been shown to act in uterine and vulval development. Here, we provide evidence that C. elegans fos-1 and jun-1 control ovulation, a tightly regulated rhythmic program in animals. Knockdown of fos-1 or jun-1 blocks dilation of the distal spermathecal valve, a critical step for the entry of mature oocytes into the spermatheca for fertilization. Furthermore, fos-1 and jun-1 regulate the spermathecalspecific expression of plc-1, a gene that encodes a phospholipase C (PLC) isozyme that is rate-limiting for inositol triphosphate production and ovulation, and overexpression of PLC-1 rescues the ovulation defect in fos-1(RNAi) worms. Unlike fos-1, regulation of ovulation by jun-1 requires genetic interactions with eri-1 and lin-15B, which are involved in the RNA interference pathway and chromatin remodeling, respectively. At least two isoforms of jun-1 are coexpressed with fos-1b in the spermatheca, and different AP-1 dimers formed between these isoforms have distinct effects on the activation of a reporter gene. These findings uncover a novel role for FOS-1 and JUN-1 in the reproductive system and establish C. elegans as a model for studying AP-1 dimerization.

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Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis

Cited by 78 publications

References 31 publications

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

The Deubiquitinating Enzyme USP8 Promotes Trafficking and Degradation of the Chemokine Receptor 4 at the Sorting Endosome

Caenorhabditis elegans FOS-1 and JUN-1 Regulateplc-1Expression in the Spermatheca to Control Ovulation

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