Dong Q et al. (1995) International C. elegans Meeting "FISHING FOR PROTEINS WHICH INTERACT WITH LIN-12 & GLP-1"

Dong Q, Greenwald IS

[International C. elegans Meeting, 1995]

lin-12 belongs to a large family of transmembrane receptor proteins including glp-1 in C. elegans, Notch in Drosophila, and TAN-1, int-3 of mammals. All members of the lin-12/Notch family have multiple EGF repeats in their extracellular domains and cdc10/SWI6 (ankyrin) repeats in their intracellular domains. There is also evidence that lin-12/Notch proteins share conserved ligands and effectors. Given that the cdc10/SWI6 repeats are known to be involved in protein-protein interactions, it is likely that the cdc10/SWI6 repeats of lin-12 and glp-1 mediate interaction with downstream effectors. We have used the yeast two hybrid system and screened a miniature fusion library made by our lab and a comprehensive fusion library made by Dr. Robert Barstead. In the process of making GAL-4 DNA binding fusion constructs, we found that the intact intracellular domain of lin-12 has the activation activity. By truncating the C terminal portion of lin-12 intracellular domain, leaving the cdc10/SWI6 repeats intact, we were able to make a construct without activation activity. We also made an equivalent construct of the glp-1 intracellular domain. By selecting their ability to grow on 3-AT plate (His+ selection) and their ability to turn blue on beta-gal filter assay when the lin-12 bait was presented, we were able to identify 51 potential candidates. Judging by their ability to interact with both lin-12 and glp-1 intracellular domain baits, the strength of the interaction on filter assay and partial sequencing analysis, we were able to narrow this group down to 13 candidates. We have mapped all the candidates and are doing sequence analysis on all of the inserts. Based on their map position, we are also using genomic phage clones from the genes found in our screen in transformation rescue experiments. Out of the 13 candidates, two correspond to known genes, emb-5 and skn-1. We are examining existing emb-5 and skn-1 mutants for phenotypes associated with mutations in lin-12 or glp-1, and examining the phenotypes of various double mutants for genetic interactions. We will report our progress at the meeting (see also the abstract of Hubbard & Greenwald).

Dong M-Q et al. (1998) East Coast Worm Meeting "Three G Protein Inhibitors Stimulate Egg Laying in C. elegans"

Koelle MR, Dong M-Q

[East Coast Worm Meeting, 1998]

RGS proteins (regulators of G protein signaling) inhibit G proteins signaling by directly converting active G proteins (GTP-bound) into their inactive GDP-bound forms. Eleven genes with RGS homology have been identified in the C. elegans genome. We found that only three of them, egl-10, C05B5.7, and F16H9.1, stimulate egg laying when they are overexpressed, suggesting that these three genes may somehow function together to control egg laying. EGL-10 has previously been shown to inhibit GOA-1, a G protein alpha subunit, which in turn inhibits egg laying1. Our current interest is in understanding the roles of C05B5.7 and F16H9.1 in the regulation of egg laying, and in understanding the relationship among the three RGS genes and goa-1. We have obtained cDNAs for C05B5.7 and F16H9.1, which encode the most similar pair of RGS proteins in C. elegans (77% identity in the RGS domain and 64% identity overall). We examined the expression patterns of C05B5.7 and F16H9.1 by injecting promoter::GFP constructs into worms. Expression of C05B5.7::GFP was found in most or all neurons, resembling the expression patterns of both egl-10 and goa-1. F16H9.1::GFP was expressed in a large subset of neurons and in the uterine muscles. We are analyzing a C05B5.7 knock-out strain obtained for us by NemaPharm. So far we have not found any egg-laying defects in the C05B5.7 single mutant. However, preliminary data suggest that the C05B5.7 mutation enhances the egg-laying defect of n480, a weak egl-10 allele. We also found that overexpression of C05B5.7 from an integrated transgene array rescued the egg-laying defect of an egl-10 null mutant. We are trying to obtain F16H9.1 mutants to complete our studies. As C05B5.7 and F16H9.1 may be functionally redundant, given the close similarity in their protein sequences and expression patterns, we may need to knock out both genes to understand their functions. Biochemical analysis of the three RGS proteins are in progress. Our short-term goal is to examine whether and how well C05B5.7 and F16H9.1 proteins stimulate the GTPase activity of GOA-1 in vitro as compared to EGL-10. This may help explain why there are multiple RGS genes involved in the signaling pathway that regulates egg laying. 1. Koelle, M.R. and Horvitz, H.R. (1996) Cell 84, 115-125

Dong M-Q et al. (2000) Genes Dev "Multiple RGS proteins alter neural G protein signaling to allow C. elegans to rapidly change behavior when fed."

Koelle MR, Chase DL, Patikoglou GA, Dong M-Q

[Genes Dev, 2000]

Regulators of G protein signaling (RGS proteins) inhibit heterotrimeric G protein signaling by activating G protein GTPase activity. Many mammalian RGS proteins are expressed in the brain and can act in vitro on the neural G protein G(o), but the biological purpose of this multiplicity of regulators is not clear. We have analyzed all 13 RGS genes in Caenorhabditis elegans and found that three of them influence the aspect of egg-laying behavior controlled by G(o) signaling. A previously studied RGS protein, EGL-10, affects egg laying under all conditions tested. The other two RGS proteins, RGS-1 and RGS-2, act as G(o) GTPase activators in vitro but, unlike EGL-10, they do not strongly affect egg laying when worms are allowed to feed constantly. However, rgs-1; rgs-2 double mutants fail to rapidly induce egg-laying behavior when refed after starvation. Thus EGL-10 sets baseline levels of signaling, while RGS-1 and RGS-2 appear to redundantly alter signaling to cause appropriate behavioral responses to food.

Arroyo A et al. (2006) FEBS Lett "Coenzyme Q is irreplaceable by demethoxy-coenzyme Q in plasma membrane of Caenorhabditis elegans."

Arroyo A, Navas P, Gavilan A, Padilla S, Ruiz-Ferrer M, Santos-Ocana C, Rodriguez-Aguilera JC

[FEBS Lett, 2006]

A procedure was developed to isolate fractions enriched in plasma membrane from Caenorhabditis elegans. Coenzyme Q(9) (Q(9)) was found in plasma membrane isolated from either wild-type or long-lived qm30 and qm51 clk-1 mutant strains of Caenorhabditis elegans, along with dietary coenzyme Q(8) (Q(8)) and the biosynthetic intermediate demethoxy-Q(9) (DMQ(9)). NADH was able to reduce both Q(8) and Q(9), but not DMQ(9). Our results indicate that DMQ(9) cannot achieve the same redox role of Q(9) in plasma membrane, suggesting that proportion of all these Q isoforms in plasma membrane must be an important factor in establishing the clk-1 mutant phenotype.

Jonassen T et al. (2003) J Biol Chem "Reproductive fitness and quinone content of Caenorhabditis elegans clk-1 mutants fed coenzyme Q isoforms of varying length."

Larsen PL, Clarke CF, Jonassen T, Davis DE

[J Biol Chem, 2003]

Caenorhabditis elegans clk-1 mutants lack coenzyme Q(9) and accumulate the biosynthetic intermediate demethoxy-Q(9). A dietary source of ubiquinone (Q) is required for larval growth and development of the gonad and germ cells. We considered that uptake of the shorter Q(8) isoform present in the Escherichia coli food may contribute to the Clk phenotypes of slowed development and reduced brood size observed when the animals are fed Q-replete E. coli. To test the effect of isoprene tail length, N2 and clk-1 animals were fed E. coli engineered to produce Q(7), Q(8), Q(9), or Q(10). Wild-type nematodes showed no change in reproductive fitness regardless of the Q(n) isoform fed. clk-1(e2519) fed the Q(9) diet showed increased egg production; however, this diet did not improve reproductive fitness of the clk-1(qm30) animals. Furthermore, animals with the more severe clk-1(qm30) allele become sterile and their progeny inviable when fed Q(7)-containing bacteria. The content of Q(7) in the mitochondria of clk-1 animals was decreased relative to Q(8), suggesting less effective transport of Q(7) to the mitochondria, impaired retention, or decreased stability. Additionally, regardless of E. coli diet, clk-1(qm30) animals contain a dysfunctional dense form of mitochondria. The gonads of clk-1(qm30) worms fed Q(7)-containing food were severely shrunken and disordered. The differential fertility of clk-1 mutant nematodes fed Q isoforms may result from changes in Q localization, altered recognition by Q-binding proteins, and/or potential defects in mitochondrial function resulting from the mutant CLK-1 polypeptide itself.

Walker MW et al. (2005) J Biomol Screen "Use of Caenorhabditis elegans G{alpha}q chimeras to detect G-protein-coupled receptor signals."

Smith KE, Branchek TA, Walker MW, Jones KA, Gerald C, Tamm J, Zhong H, Vaysse P

[J Biomol Screen, 2005]

G-protein-coupled receptors (GPCRs) activate heterotrimeric G-proteins (G(i)-, G(s)-, G(q)-, or G(12)-like) to generate specific intracellular responses, depending on the receptor/G-protein coupling. The aim was to enable a majority of GPCRs to generate a predetermined output by signaling through a single G-protein-supported pathway. The authors focused on calcium responses as the output, then engineered Galpha(q) to promote promiscuous receptor interactions. Starting with a human Galpha(q) containing 5 Galpha(z) residues in the C-terminal receptor recognition domain (hGalpha(q/z5)), they evaluated agonist-stimulated calcium responses for 33 diverse GPCRs (G(i)-, G(s)-, and G(q)-coupled) and found 20 of 33 responders. In parallel, they tested Caenorhabditis elegans Galpha(q) containing 5 or 9 C-terminal Galpha(z) residues (cGalpha(q/z5), cGalpha(q/z9)). Signal detection was enhanced with cGalpha(q/z5) and cGalpha(q/z9) (yielding 25/33 and 26/33 responders, respectively). In a separate study of Galpha(s)-coupled receptors, the authors compared hGalpha(q/s5) versus hGalpha(q/s9), cGalpha(q/s9), andcGalphaq/s21 and observed optimal function with cGalpha(q/s9). Cotransfection of an engineered Galpha(q) "cocktail" (cGalpha(q/z5) plus cGalpha(q/s9)) provided a powerful and efficient screening platform. When the chimeras included N-terminal myristoylation sites (to promote membrane localization), calcium responses were sustained or improved, depending on the receptor. This approach toward a "universal functional assay" is particularly useful for orphan GPCRs whose signaling pathways are unknown.

Gaur R et al. (2007) J Biosci "Diet-dependent depletion of queuosine in tRNAs in Caenorhabditis elegans does not lead to a developmental block."

Gaur R, Tuck S, Bjork GR, Varshney U

[J Biosci, 2007]

Queuosine (Q),a hypermodified nucleoside,occurs at the wobble position of transfer RNAs (tRNAs)with GUN anticodons.In eubacteria,absence of Q affects messenger RNA (mRNA)translation and reduces the virulence of certain pathogenic strains.In animal cells,changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance,cell proliferation and tumour growth but the function of Q in animals is poorly understood.Animals are thought to obtain Q (or its analogues) as a micronutrient from dietary sources such as gut micro flora.However,the difficulty of maintaining animals under bacteria-free conditions on Q-deficient diets has severely hampered the study of Q metabolism and function in animals.In this study,we show that as in higher animals, tRNAs in the nematode Caenorhabditis elegans are modified by Q and its sugar derivatives.When the worms were fed on Q-deficient Escherichia coli ,Q modification was absent from the worm tRNAs suggesting that C.elegans lacks a de novo pathway of Q biosynthesis.The inherent advantages of C.elegans as a model organism,and the simplicity of conferring a Q-deficient phenotype on it make it an ideal system to investigate the function of Q modification in tRNA.

Jonassen T et al. (2002) J Biol Chem "Development and fertility in Caenorhabditis elegans clk-1 mutants depend upon transport of dietary coenzyme Q8 to mitochondria."

Marbois BN, Jonassen T, Faull KF, Clarke CF, Larsen PL

[J Biol Chem, 2002]

The Caenorhabditis elegans clk-1 mutants lack coenzyme Q, and instead accumulate the biosynthetic intermediate demethoxy-Q(9) (DMQ(9)). clk-1 animals grow to reproductive adults, albeit slowly, if supplied with Q(8)-containing Escherichia coli. However, if Q is withdrawn from the diet, clk-1 animals either arrest development as young larvae or become sterile adults depending upon the stage at the time of the withdrawal. To understand this stage-dependent response to a Q-less diet, the quinone content was determined during development of wild-type animals. The quinone content varies in the different developmental stages in wild-type fed Q(8)-replete E. coli. The amounts peak at the second larval stage, which coincides with the stage of arrest of clk-1 larvae fed a Q-less diet from hatching. Levels of the endogenously synthesized DMQ(9) are high in the clk1(qmM30)-arrested larvae and sterile adults fed Q-less food. Comparison of quinones from animals fed a Q-replete or a Q-less diet establishes that the Q(8) present is assimilated from the E. coli. Furthermore, this E. colispecific Q(8) is present in mitochondria isolated from fertile clk-1(qm30) adults fed a Q-replete diet. These results suggest that the uptake and transport of dietary Q(8) to mitochondria prevent the arrest and sterility phenotypes of clk-1 mutants and that DMQ is not functionally equivalent to Q.

Santos-Ocana C et al. (2002) J Biol Chem "Uptake of exogenous coenzyme Q and transport to mitochondria is required for bc1 complex stability in yeast coq mutants."

Clarke CF, Santos-Ocana C, Padilla S, Navas P, Do TQ

[J Biol Chem, 2002]

Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery in Saccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q(6) by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-less coq7 mutants derived from four distinct laboratory yeast strains. Q(6) supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the four coq7 mutant strains. Following culture in medium containing dextrose, the added Q(6) was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q(6) in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q(6). These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q(6) into the plasma membrane. The two coq7 mutant strains rescued by Q(6) supplementation for growth on ethanol contained mitochondrial Q(6) levels similar to wild type. However, the content of Q(6) in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q(6), succinate-cytochrome c reductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q(6) content, succinate-cytochrome c reductase activity, and steady state levels of the cytochrome c(1) polypeptide. These studies show that transport of extracellular Q(6) to the mitochondria operates in yeast but is strain-dependent. When Q biosynthesis is disrupted in yeast strains with defects in the intracellular transport of exogenous Q, the bc(1) complex is unstable. These results indicate that delivery of exogenous Q(6) to mitochondria is required fore activity and stability of the bc(1) complex in yeast coq mutants.

Saiki R et al. (2008) Aging Cell "Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q."

Saiki R, Clarke CF, Furukawa S, Larsen PL, Bixler T, Lunceford AL, Lee W, Dang P

[Aging Cell, 2008]

Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. C. elegans fed E. coli devoid of Q(8) have a significant life span extension when compared to C. elegans fed a standard "Q-replete"E. coli diet. Here we examine possible mechanisms for the life span extension caused by the Q-less E. coli diet. A bioassay for Q uptake shows that a water-soluble formulation of Q(10) is effectively taken up by both clk-1 mutant and wild-type nematodes, but does not reverse life span extension mediated by the Q-less E. coli diet, indicating that life span extension is not due to the absence of dietary Q per se. The enhanced longevity mediated by the Q-less E. coli diet cannot be attributed to dietary restriction, different Qn isoforms, reduced pathogenesis or slowed growth of the Q-less E. coli, and in fact requires E. coli viability. Q-less E. coli have defects in respiratory metabolism. C. elegans fed Q-replete E. coli mutants with similarly impaired respiratory metabolism due to defects in complex V also show a pronounced life span extension, although not as dramatic as those fed the respiratory deficient Q-less E. coli diet. The data suggest that feeding respiratory incompetent E. coli, whether Q-less or Q-replete, produces a robust life extension in wild-type C. elegans. We believe the fermentation-based metabolism of the E. coli diet is an important parameter of C. elegans longevity.