KEL-8 is a substrate receptor for CUL3-dependent ubiquitin ligase that regulates synaptic glutamate receptor turnover

Abstract

The regulated localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPARs) to synapses is an important component of synaptic signaling and plasticity. Regulated ubiquitination and endocytosis determine the synaptic levels of AMPARs, but it is unclear which factors conduct these processes. To identify genes that regulate AMPAR synaptic abundance, we screened for mutants that accumulate high synaptic levels of the AMPAR subunit GLR-1 in Caenorhabditis elegans. GLR-1 is localized to postsynaptic clusters, and mutants for the BTB-Kelch protein KEL-8 have increased GLR-1 levels at clusters, whereas the levels and localization of other synaptic proteins seem normal. KEL-8 is a neuronal protein and is localized to sites adjacent to GLR-1 postsynaptic clusters along the ventral cord neurites. KEL-8 is required for the ubiquitin-mediated turnover of GLR-1 subunits, and kel-8 mutants show an increased frequency of spontaneous reversals in locomotion, suggesting increased levels of GLR-1 are present at synapses. KEL-8 binds to CUL-3, a Cullin 3 ubiquitin ligase subunit that we also find mediates GLR-1 turnover. Our findings indicate that KEL-8 is a substrate receptor for Cullin 3 ubiquitin ligases that is required for the proteolysis of GLR-1 receptors and suggest a novel postmitotic role in neurons for Kelch/CUL3 ubiquitin ligases.

Figure 1.

KEL-8 regulates GLR-1 abundance in dendrites. GLR-1::GFP (A and B), SNB-1::GFP (C and D), UNC-43::GFP (E and F), and LIN-10::GFP (G and H) fluorescence was observed along the ventral cord dendrites of wild-type nematodes (A, C, E, and G) or kel-8 mutants (B, D, F, and H). Whereas wild-type animals have small clusters of GLR-1::GFP (100%; n = 20), most kel-8 mutants have large (>2-μm) clusters of GLR-1::GFP (95%; n = 20). Bar, 5 μm. The mean cluster area (I) and the mean number of clusters per 10 μm (J) of ventral cord length is plotted for adult nematodes of the given genotype and expressing the transgene indicated beneath the graph. White bars indicate wild-type animals, whereas black bars indicate kel-8 mutants. AU, arbitrary units. Error bars are SEM for all graphs. ***p < 0.0001 and **p < 0.001 compared with wild type expressing the corresponding transgene by Student's t test. n = 15–20 animals for each genotype–transgene combination.

Figure 2.

KEL-8 encodes a member of the Kelch Superfamily. (A) The predicted intron/exon gene structure of kel-8 based on cDNA sequence is shown at top. Black boxes indicate coding sequences, whereas white boxes indicate untranslated regions. At bottom is the predicted protein domain structure, including the BTB, BACK, and six Kelch repeats. Amino acid identities and similarities to human Kelch-like 8 (KHL8) for each domain are shown. The molecular nature of the od38 mutation is indicated. (B) Phylogenetic tree for KEL-8 and various Kelch proteins in the human and C. elegans genomes. KEL-8 is most similar to human Kelch-like 8. (C) Amino acid alignment of KEL-8, human KHL8, and Drosophila KELCH. Black highlighting indicates identities, and gray highlighting indicates similarities. Overlines indicate specific protein domains. (D) Fluorescence from GLR-1::GFP in the ventral cord bundle of kel-8 mutants. (E) kel-8 mutants rescued with genomic cosmid W02G9 containing the kel-8 locus. (F) kel-8 mutants rescued cell autonomously with a P_glr-1::kel-8 transgene containing wild-type kel-8 cDNA fused to the glr-1 promoter. Bar, 5 μm.

Figure 3.

KEL-8 negatively regulates GLR-1 function. (A) The mean nose-touch sensitivity (percentage of 10 total trials per animal in which the animal reversed direction upon forward collision with an eyelash) is plotted for the given genotype. WT, wild-type animals. (B) The mean spontaneous reversal frequency (number of reversals per minute over a 5-min period) is plotted for the given genotype. Error bars are SEM for both graphs. **p < 0.01 for comparisons to wild type using analysis of variance (ANOVA) and Dunnett's multiple comparison test. n = 20 animals for each genotype.

Figure 4.

KEL-8 is expressed in neurons and localized to clusters in the ventral cord dendrites. KEL-8::GFP (A, D, G, and J) and GLR-1::RFP (B, E, H, and K) fluorescence were observed throughout the entire animal (A–C), along ventral cord neurites (D–F), along lateral head ganglia (G–I), and in the lumbar ganglia of the tail (J–L). KEL-8::GFP is observed in the nerve ring (NR), lateral and ventral ganglia (L&VG), the ventral nerve cord (VNC), and the lumbar ganglia (LG) of the tail (merged image in C). Intestinal autofluorescence (IA), which is nonspecific and does not indicate expression from either transgene, is observed throughout the midbody. Along the ventral cord, KEL-8::GFP is localized to clusters adjacent to clusters of GLR-1::RFP along the ventral cord (merged image in F). KEL-8::GFP and GLR-1::RFP are expressed in the same lateral and lumbar cells bodies (merged images in I and L, respectively; cell identities are indicated). (M and N) GLR-1::CFP fluorescence was observed in kel-8 mutants (M) or kel-8 mutants (N) rescued cell autonomously with a P_glr-1::kel-8::yfp transgene containing wild-type kel-8 cDNA fused in frame to YFP. The KEL-8::YFP chimeric protein functions to rescue the kel-8 mutant phenotype in these neurons. Bars, 20 μm (A–C), 5 μm (D–L), and 10 μm (M and N).

Figure 5.

KEL-8 is required for ubiquitin-mediated turnover of GLR-1. (A–G) GLR-1::GFP fluorescence was observed along ventral cord dendrites of wild-type nematodes (A), wild-type nematodes carrying a transgene that overexpresses MUb (B), unc-11 mutants (C), unc-11 mutants overexpressing MUb (D), kel-8 mutants (E), kel-8 mutants overexpressing MUb (F), and unc-11 kel-8 double mutants (G). Overexpression of ubiquitin results in decreased GLR-1 levels, whereas unc-11 and kel-8 mutations result in increased levels of GLR-1. Moreover, kel-8 mutations, like unc-11 mutations, partially block the downregulation of GLR-1 by overexpression of ubiquitin. Double mutants for kel-8 and unc-11 accumulate GLR-1 in a similar manner to kel-8 mutants. Bar, 5 μm. (H) Quantification of the GLR-1 cluster area and number (per 10 μm of neurite length) comparing unc-11 and kel-8 single and double mutants. (I) Quantification of the GLR-1 cluster area and number comparing kel-8 in the presence or absence of overexpressed ubiquitin (MUb transgene). Error bars are SEM for all graphs. *p < 0.05 and ***p < 0.001 for intergenotype comparisons using ANOVA and Bonferroni multiple comparison test. n = 20 animals for each genotype.

Figure 6.

KEL-8 interacts with CUL-3 Cullin. (A) GST::CUL-3 and FLAG::KEL-8 were cotransfected into COS-7 cells, which were solubilized in RIPA buffer. GST affinity chromatography was performed, and bound proteins were detected by Western blotting (WB) with an antisera that recognizes both GST and KEL-8. GST::CUL-3 but not GST alone pulls down KEL-8 protein. GST::CUL-3(H2), which reduces the affinity of CUL-3 for other BTB proteins, pulls down reduced amounts of KEL-8. (B) Immunoprecipitations were performed with anti-FLAG antisera, and coimmunoprecipitated proteins were detected by Western blotting with an anti-GST antisera. FLAG::KEL-8 coprecipitates GST::CUL-3 but not GST alone. Reduced amounts of GST::CUL-3(H2) (only detectable with significantly longer chemiluminescence exposures than the blot shown) are coprecipitated compared with wild-type GST::CUL-3. For both A and B, arrowheads indicate the specific pulled down proteins. Brackets above the gels indicate the contransfected constructs. “Load” indicates 2.5% of the original lysate. Similar results were observed in six separate transfection experiments. (C) AH109 yeast expressing CUL-3 fused to Gal4 DNA binding domain (DB CUL-3) and KEL-8 fused to an activation domain (AD KEL-8) are able to support growth on two independent yeast two-hybrid reporter genes (HIS3 and ADE2). The DB CUL-3 and AD KEL-8 constructs, present individually in AH109 and streaked out in separate quadrants, are unable to support growth. (D–H) GLR-1::GFP clusters were visualized in wild-type nematodes (D), nematodes expressing full-length CUL-3 protein (E), nematodes expressing amino-terminal residues 1–500 (F), nematodes expressing carboxy-terminal residues 501–777 (G), and rfl-1 mutants (H). Expression of either CUL-3(1-500) or CUL-3(501-777), or a mutation in the Nedd8-activating enzyme RFL-1 results in the accumulation of GLR-1. Bar, 5 μm.