Figure S1. USP-46 is expressed in several tissues including the nervous system

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1 Supplemental Figure legends Figure S1. USP-46 is expressed in several tissues including the nervous system Transgenic animals expressing a transcriptional reporter (P::GFP) were imaged using epifluorescence (1) and Nomarski optics (2). P::GFP is expressed in the pharynx (A), in many neurons in the head (A), in the nerve ring (B), in body wall muscle (C), in the intestine (D), and in vulval muscles (E). The asterisks (*) in (D) and (E) mark the position of the vulva. (F-H) USP-46 is expressed in GLR-1 expressing head neurons. Transgenic animals co-expressing a transcriptional reporter (P::GFP)(F), and a glr-1 transcriptional reporter (Pglr-1::dsRED)(G) were imaged. (H) Merged image of (F) and (G). (F2-H2) White dotted lines outline the neuronal cell bodies for clarity. The neuron on the far left expresses (F2) but not glr-1 (G2). Neurons co-expressing and glr-1 are indicated by white asterisks (*) in (H2). Figure S2. Analysis of Multiple Synaptic Markers in mutant animals (A-B) Representative images of MAGI-1::YFP puncta in the anterior ventral nerve cords of wild type (A) and (ok2232)(b) L4 larvae expressing MAGI-1::YFP. The asterisk marks a neuronal cell body. (C) Quantification of MAGI-1::YFP puncta densities (puncta/10 m) for the strains pictured in (A-B). Shown are the means and standard errors (SEM) for n = 24 wild type and n = 24 (ok2232). (D-E) Representative images of LIN-10::GFP puncta in the anterior ventral nerve cords of wild type (D) and (ok2232)(e) L4 larvae expressing LIN-10::GFP. (F) Quantification of LIN-10::GFP puncta densities (puncta/10 m) for the strains pictured in (D-E). Shown are the means and standard errors (SEM) for n = 23 wild type and n = 18 (ok2232). 1

2 (G-H) Representative images of SOL-1::YFP puncta in the anterior ventral nerve cords of wild type (G) and (ok2232)(h) L4 larvae expressing SOL-1::YFP. (I) Quantification of SOL-1::YFP puncta densities (puncta/10 m) for the strains pictured in (G-H). Shown are the means and standard errors (SEM) for n = 13 wild type and n = 16 (ok2232). Figure S3. In vitro binding assay of GST-GLR-1 and USP-46 USP-46 or the last three PDZ domains of MAGI-1 were in vitro translated and labeled with 35 S-methionine. The labeled proteins were incubated with GST, lanes 2 and 5, or GST fused to the C-terminus of GLR-1 (GST-GLR-1C), lanes 3 and 6, and subsequently pulled down with glutathione sepharose beads. Lanes 1 and 4 show 10% of the input of 35 S-labeled protein. Reactions were analyzed by SDS-PAGE and Coomassie staining (lower panel) and fluorography (upper panel). Arrows indicate the position of GST alone or GST-GLR-1C (lower panel). Figure S4. Analysis of ubiquitinated GLR-1 (A) Control immunoprecipitation experiment detecting ubiquitinated GLR-1::GFP from membrane preparations of mixed stage wild type animals expressing GLR-1::GFP (nuis24). Total GLR-1::GFP was first immunoprecipitated using polyclonal anti-gfp antibodies (1 st IP). This was followed by a second, sequential immunoprecipitation of ubiquitin-glr-1 conjugates using polyclonal anti-ubiquitin antibodies (2 nd IP) (top panel, lanes 1 and 2). In this experiment, purified, free ubiquitin monomers (+Ub) or an equal concentration of bovine serum albumin (BSA) (-Ub) were preincubated with the antiubiquitin polyclonal antibodies prior to use in the 2 nd IP step. Excess free ubiquitin or excess BSA was also included in the corresponding 2 nd IP. Western blot analyses were used to detect immunoprecipitated proteins from each IP. Immunoprecipitated GLR- 2

3 1::GFP from the 1 st IP was detected with monoclonal anti-gfp antibodies (lower blot). Ubiquitin-GLR-1::GFP conjugates immunoprecipitated in the 2 nd IP were detected with monoclonal anti-gfp antibodies (upper blot). Note that the 3 rd lane contains Total membranes and indicates that non-ubiquitinated GLR-1::GFP runs below the apparent molecular weight of the Ub-GLR-1::GFP bands. (B) Control Immunoprecipitation experiment detecting ubiquitinated GLR-1::GFP in membranes prepared from mixed stage populations of wild type animals expressing GLR-1::GFP (nuis25). Total GLR-1::GFP was first immunoprecipitated using polyclonal anti-gfp antibodies (1 st IP). This was followed by a second, sequential immunoprecipitation of ubiquitin-glr-1 conjugates using polyclonal anti-ubiquitin antibodies (2 nd IP). Western blot analyses were used to detect immunoprecipitated proteins from each IP. Immunoprecipitated GLR-1::GFP from the 1 st IP and 2 nd IP were detected with monoclonal anti-gfp antibodies (left blot). Ubiquitin-GLR-1::GFP conjugates immunoprecipitated in the 2 nd IP were detected with monoclonal antiubiquitin antibodies (right blot). 120 times more material was loaded for the blots of the 2 nd IPs than for the anti-gfp blot of the 1 st IP. Figure S5. In vitro DUB activity of GST-USP-46 (A) Recombinant UCH-L3 (positive control) or GST-USP-46 were incubated with the inhibitor HA-tagged vinyl methyl ester (HA-Ub-VME) to covalently label active DUB species. Reaction with HA-Ub-VME causes an increase in the apparent molecular weight of the DUB (arrows). Reactions were analyzed by SDS-PAGE followed by either Coomassie staining (left panel) or Western blotting with anti-ha antibodies to detect HAtagged DUB species (right panel). About 50% of UCH-L3 is active and covalently labeled by HA-Ub-VME as detected by the anti-ha Western blot and the mobility shift in the coomaasie stained gel (lower arrows). In contrast, only a small percentage of GST- 3

4 USP-46 is active and covalently modified by HA-Ub-VME (top arrows). The asterisks indicate the apparent molecular weight of unreacted, inactive GST-USP-46. MW (KDa) markers are shown. (B) USP-46 deubiquitination assay. Recombinant GST-USP-46 or catalytically inactive GST-USP-46(C>A) were incubated with GLR-1::GFP isolated from membrane fractions of mixed stage wild type worms expressing GLR-1::GFP in DUB reaction buffer. The relative amounts of ubiquitinated GLR-1::GFP was subsequently determined. Total GLR-1::GFP was first immunoprecipitated using polyclonal anti-gfp antibodies (1 st IP), followed by a second, sequential immunoprecipitation of ubiquitin- GLR-1 conjugates using polyclonal anti-ubiquitin antibodies (2 nd IP). Western blot analyses with monoclonal anti-gfp antibodies were used to detect immunoprecipitated GLR-1::GFP from the 1 st IP (left panel) and Ubiquitin-GLR-1::GFP conjugates immunoprecipitated in the 2 nd IP (right panel). 120 times more material was loaded for the anti-gfp blot of the 2 nd IP than for the anti-gfp blot of the 1 st IP. Numbers listed below the right panel indicate the normalized ratios of the amount of ubiquitinated-glr-1 (right panel) to the amount of total immunoprecipitated GLR-1::GFP (left panel) (Ub- GLR-1/GLR-1). MW (kda) markers are indicated. (C) Graph representing the average ratio of Ub-GLR-1/GLR-1 normalized to GST-USP-46(C>A) treated samples (n=3). SEM shown. Figure S6. Quantification of Puncta Widths for experiments shown in Figures 5 and 6 (A) Quantification of GLR-1::GFP puncta widths for the strains pictured in Figure 5A-D. Shown are the means and standard errors (SEM) for n = 59 wild type, n = 41 (ok2232), n = 20 unc-11 AP180(e47), and n = 24 unc-11 AP180; animals. Values that differ significantly from wild type (Student s t test) are denoted by asterisks (*) above each bar. All significant differences are indicated as follows: * p 0.01, ** p

5 (B) Quantification of GLR-1::GFP puncta widths for the strains pictured in Figure 6A-D. Shown are the means and standard errors (SEM) for n = 26 wild type, n = 19 (ok2232), n = 22 vps-4(dn), and n = 26 vps-4(dn); animals. Values that differ significantly from wild type (Student s t test) are denoted by asterisks (*) above each bar. All significant differences are indicated as follows: # p 0.02, * p 0.01, ** p n.s. denotes no significant difference between the indicated strains (p > 0.05). Figure S7. USP-46 localization in the VNC (A-B) Representative images of mcherry::usp-46 (A) or GLR-1::GFP (B) expressed under the control of the glr-1 promoter in the anterior ventral nerve cords of wild type L4 larvae. (C) Merged image of (A) and (B). Arrowheads indicate mcherry::usp-46 puncta that do not co-localize with GLR-1::GFP punta. Arrows indicate mcherry::usp-46 puncta that exhibit some overlap. Figure S8. USP-46 localizes to an endosomal compartment but not the ER (A) Representative images of the ER marker KDEL::venus (A) and mcherry::usp-46 (B) expressed under the control of the glr-1 promoter in PVC interneuron cell bodies. (C) Merged image of (A) and (B). (D-E) Endo H assay. (D) GLR-1::GFP containing membranes from wild type or (ok2232) mutant animals were either left untreated (lanes 1 and 4), digested with Endo H (lanes 2 and 5) or digested with PNGase F (lanes 3 and 6). Western blot analyses with anti-gfp antibodies were used to detect GLR- 1::GFP. Endo H-sensitive (Endo H-S) and Endo H-resistant (Endo H-R) GLR-1::GFP bands are indicated. (E) Quantitation of the percentage of Endo H-sensitive GLR- 1::GFP averaged from three experiments. Errors bars show standard error of the mean (SEM). (F-G) Representative images of mcherry::usp-46 (F) or Venus::RAB-5 (G) puncta expressed under the control of the glr-1 promoter in the anterior ventral nerve 5

6 cords of wild type L4 larvae. (H) Merged image of (F) and (G). Arrows indicate mcherry::usp-46 puncta that are co-localized with Venus::RAB-5 puncta. (I-J) Representative images of mcherry::usp-46 (I-J) or Venus::RAB-5 (K-L) puncta expressed under the control of the glr-1 promoter in the PVC neuron cell bodies of wild type animals. (M) Merged image of (I) and (K). (N) Merged image of (J) and (L). (O) Analysis of ubiquitinated GLR-1 in animals expressing vps-4(dn). Representative immunoprecipitation experiments showing the relative amounts of ubiquitinated GLR- 1::GFP in membranes prepared from mixed stage populations of wild type or mutant animals expressing GLR-1::GFP and vps-4(dn). Total GLR-1::GFP was first immunoprecipitated using polyclonal anti-gfp antibodies (1 st IP), followed by a second, sequential immunoprecipitation of ubiquitin-glr-1 conjugates using polyclonal antiubiquitin antibodies (2 nd IP). Western blot analyses with monoclonal anti-gfp antibodies were used to detect immunoprecipitated GLR-1::GFP from the 1 st IP (left panel) and Ubiquitin-GLR-1::GFP conjugates immunoprecipitated in the 2 nd IP (right panel). 120 times more material was loaded for the anti-gfp blot of the 2 nd IP than for the anti-gfp blot of the 1 st IP. Numbers listed below the right panel indicate the normalized ratios of the amount of ubiquitinated-glr-1 (right panel) to the amount of total immunoprecipitated GLR-1::GFP (left panel) (Ub-GLR-1/GLR-1). MW (kda) markers are indicated. Similar results were observed in three independent immunoprecipitation experiments performed on a total of two membrane preparations per strain. 6

7 A1 pharynx A2 B1 B2 head neurons nerve ring C1 D1 intestine C2 body wall muscle D2 * * E1 vulva muscle E2 * * F1 G1 H1 F2 G2 H2 5 m * P::GFP Pglr-1::dsRED Merge * * *

8 MAGI-1::YFP A B * 10 m C D ensity (puncta/10 m) WT LIN-10::GFP D E 10 m F D ensity (puncta/10 m) WT G H SOL-1::YFP 10 m I D ensity (puncta/10 m) WT

9 MAGI-1 (3PDZ) USP-46 Input GST GST-GLR-1C Input GST GST-GLR-1C 35 S-MAGI-1 (3PDZ) 35 S-USP-46 MW MW Coomassie stain GST-GLR-1C GST

10 A -Ub +Ub Total GLR-1::GFP Ub-GLR-1 1st IP: -GFP 2nd IP: -Ub Blot: -GFP GLR-1::GFP 1st IP: -GFP Blot: -GFP B Total 1st IP: -GFP 2nd IP: -Ub 2nd IP: -Ub GLR-1::GFP Ub-GLR Blot: -GFP Blot: -Ub

11 A HA-Ub-VME HA-Ub-VME UCHL3 GST- USP46 UCHL3 GST- USP * * Labelled GST-USP Labelled UCHL Coomassie stained gel HA Western blot (HA-Ub conjugates) B C 216 GLR st IP: -GFP WB: -GFP GST-USP-46 C>A WT Ub-GLR-1 GLR-1 Ratio: 2nd IP: -Ub WB: -GFP GST-USP-46 C>A WT Ub-GLR-1 Ub-GLR-1/GLR-1 ratio GST-USP-46 (C>A) GST-USP-46 (WT)

12 A Puncta Width ( m) B Puncta Width ( m) ** ** ** ** # ** n.s. 0 unc-11 AP180 unc-11 AP180; 0 vps-4(dn) vps-4(dn);

13 A mcherry::usp-46 B GLR-1::GFP C Merge 10 m

14 A B C 5 m KDEL::venus mcherry::usp-46 Merged D 200 Untreated Endo H PNGase F Untreated Endo H PNGase F E Endo H-S (%) Endo H-R Endo H-S 0 F mcherry-usp-46 I J mcherry-usp-46 mcherry-usp-46 G Venus::RAB-5 K L Venus::RAB-5 Venus::RAB-5 H Merge M 5 m N 10 m Merge Merge O 1st IP: -GFP WB: -GFP 2nd IP: -Ub WB: -GFP DN-VPS-4 DN-VPS-4; DN-VPS-4 DN-VPS-4; 216 Ub-GLR-1 GLR Ub-GLR-1 GLR-1 Ratio: