Supplemental Materials IDENTIFICATION OF TUBULIN DEGLUTAMYLASE AMONG CAENORHABDITIS ELEGANS AND MAMMALIAN CYTOSOLIC CARBOXYPEPTIDASES (CCPs) Yoshishige Kimura, Nobuya Kurabe, Koji Ikegami, Koji Tsutsumi, Yoshiyuki Konishi, Oktay Ismail Kaplan, Hirofumi Kunitomo, Yuichi Iino, Oliver E. Blacque and Mitsutoshi Setou Supplemental Methods Supplemental References Supplemental Figure Legends 1–10 Supplemental Figures 1–10 Supplemental Tables 1

Supplemental Methods Worm strains—Deletion alleles of following genes: tm3310 for ttll-4, and tm3889 for ttll-9 obtained from National BioResource Project, ok1821 for ccpp-1, ok382 for ccpp-6, and gk482 for ttll-11 obtained from C. elegans Knock-out Consortium were outcrossed 5 times. The following strains were used in this paper. Mutants: YT315: ttll-4(tm3310)III, YT432: ttll-9(tm3889)V, VC1105: ttll-11(gk482)IV, YT340: ccpp-1 (ok1821)I, YT426: ccpp-6(ok382)II, YT325: ccpp-6(ok382)II; ttll-4(tm3310)III. Transgenic strains: YT1580: ttll-4(tm3310)III; tzIs[che-2::TTLL-4::mCherry], YT400: tzEx[TTLL-4::GFP], YT371: tzEx[ttll-5::GFP], YT1610: tzEx[TTLL-9::GFP], YT1643: tzEx[ttll-11a::GFP], YT1615: tzEx[TTLL-11::GFP], YT1616: tzEx[TTLL-15::GFP], YT1574: tzEx[ccpp-1::GFP], YT395: tzEx[CCPP-1::mCherry], YT381: tzEx[CCPP-6::GFP], YT419: ttll-4(tm3310)III; otIs3[gcy-7::GFP] and YT423: ttll-4(tm3310)III; oyIs18[gcy-8::GFP]. Marker strains obtained from Caenorhabditis Genetic Center: ASEL marker OH3191: otIs3[gcy-7::GFP]V, ADL marker GR1366: mgIs42[tph-1::GFP], AFD marker PY1322: oyIs18[gcy-8::GFP], CEP marker BZ555: egIs1[dat-1::GFP] and ASK marker CX3344: kyIs53[odr-10::GFP]X. Transgenic worm strains—The PCR fusion-based GFP reporter gene was generated as described previously (1). The genomic fragment starting 1.3 kb upstream of the first Met of ttll-4 and ending at exon1, 2, and the stop codon was amplified by PCR. The GFP-unc-54 untranslated region (UTR) from pPD95.77 (a gift from A. Fire) was amplified. Other fusion reporter genes (ttll-5, -9, -11, and -15) for different fluorescent proteins (GFP and mCherry) were amplified as summarized in Supplemental Fig. 4. Germline transformation was carried out as described previously (2,3). The host strain was wild-type N2. The pRF4 plasmid (Rol) was used as a coinjection marker. The quantity of all test DNA was 30–100 ng/µl. Immunohistochemistry—The animals were fixed in modified Bouin’s fixative (400 µl of plain Bouin’s fixative, 400 µl of methanol and 0.01 ml of β-mercaptoethanol; plain Bouin’s fixative: 0.75 ml of saturated picric acid, 0.25 ml of formalin (36~38% formaldehyde solution), 0.05 ml of glacial acetic) for 30 min and immediately frozen in liquid nitrogen. Antibody incubation was performed overnight at 4°C in block solution (1 X phosphate-buffered saline [PBS], 0.5% Triton X-100, 1 mM EDTA [pH 8], 0.1% bovine serum albumin [BSA], 0.05% sodium azide, and 10% goat serum). GT335 (kindly provided by Dr. B. Eddé) was diluted 1:5000 (in Fig. 1B) or 1:2.5 × 106 (for signal quantification, as shown in Fig. 1C, and Figs 2, B and C) before incubation. The anti-GFP polyclonal antibody (MBL) was diluted 1:200. The anti-α-tubulin polyclonal antibody (MBL) was diluted 1:200. After washing with antibody buffer (4), the worms were incubated overnight in 1:500 Alexa Fluor 488- or 594-conjugated anti-mouse or anti-rabbit secondary antibody (Molecular Probes). After washing with antibody buffer, worms are analyzed by confocal microscopy FV1000 (Olympus) described in EXPERIMENTAL PROCEDURES. DNA construction—The vector che-2::TTLL-4::mCherry was constructed as follows. The che-2 promoter from the che-2::Venus expression vector, ttll-4 cDNA from yk1751h07 (a gift from Dr. Kohara, NIG), and mCherry sequence from pPD95.77-mCherry (Dr. R. Ikegami and Dr. Culotti, unpublished) were amplified and connected using a PCR-based technique.

The expression vector of CCP5 (pCMV-tag3a-CCP5) was constructed as follows. Mouse CCP5 cDNA fragment from pYX-Asc-CCP5 (Open Biosystems Inc.; accession No. BC057349) was amplified by PCR and inserted into the PstI-EcoRV site of pCMV-tag3a (Stratagene). Similarly, the expression vector of CCP6 (pCMV-tag3c-CCP6) was constructed by inserting

mouse CCP6 cDNA fragment amplified by RT-PCR from mouse testis into the EcoRV-XhoI site of pCMV-tag3c (Stratagene). The primer sequences and details of vector construction will be made available on request. In vitro deglutamylase assay—The enzyme assay in Supplemental Figure 9 was performed under two different reaction buffers: Tris-based buffer (10 mM Tris, pH 7.5, 0.1% NP-40, 10-8 M ZnCl2, 2 mM ADP) and PIPES-based buffer (10 mM PIPES-KOH pH 7.5, 100 mM NaCl, 0.1% NP-40, 10-8 M ZnCl2, 2 mM ADP). The PIPES-based buffer was used in further analyses. In vitro deglutamylase assay by bacteria—The bacterial expression vectors for C. elegans CCPP-6 and mouse CCP5 were constructed as follows. Full-length CCPP-6 and deletion mutants of CCP5 corresponding to amino acid residues 1-568 (N-terminal domain and carboxypeptidase domain) and 190-568 (only carboxypeptidase domain) were amplified by PCR and cloned into pMAL-c2X bacterial expression vector (New England Biolabs). Full-length and deletion mutants of CCP5 tagged with MBP were expressed in Escherichia coli strain BL21 (DE3) and purified with amylose resin (New England Biolabs). No deglutamylase activity was detected when any recombinants above were used (data not shown). On the other hand, full-length CCP5 was hardly solubilized. Thus, we could not detect deglutamylase activity by the recombinant expressed by bacteria probably because protein folding problem.

Cell culture and RNAi—siRNA knockdown was performed as described earlier (5). In this study, we used Stealth RNAi (Invitrogen) as the siRNA. Exogenous overexpression of siRNAs in HEK293T cells was performed as described in previous studies (5-7). Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by western blot analyses as described (8). Two-dimensional electrophoresis was performed as described (7). Mouse cortical neurons were cultured in Neurobasal medium (Invitrogen) on a poly-L-lysine-coated coverslip. At 4 days in vitro (DIV), the expression plasmids were introduced using Lipofectoamine 2000 (Invitrogen). The neurons were fixed at 6 DIV and subjected to the immunocytochemical analysis, using Mab GT335 (1:2000) and anti-β-tubulin polyclonal antibody (1:2000).

Supplement References 1. Hobert, O. (2002) Biotechniques 32, 728-730 2. Mello, C. C., Kramer, J. M., Stinchcomb, D., and Ambros, V. (1991) EMBO J 10,

3959-3970 3. Mitani, S. (1995) Dev Growth Differ 37, 551-557 4. Nonet, M. L., Staunton, J. E., Kilgard, M. P., Fergestad, T., Hartwieg, E., Horvitz, H. R.,

Jorgensen, E. M., and Meyer, B. J. (1997) J Neurosci 17, 8061-8073 5. Ikegami, K., Mukai, M., Tsuchida, J., Heier, R. L., Macgregor, G. R., and Setou, M.

(2006) J Biol Chem 281, 30707-30716 6. Ikegami, K., Horigome, D., Mukai, M., Livnat, I., Macgregor, G. R., and Setou, M.

(2008) FEBS Lett 582, 1129-1134 7. Ikegami, K., and Setou, M. (2009) FEBS Lett 583, 1957-1963 8. Ikegami, K., Heier, R. L., Taruishi, M., Takagi, H., Mukai, M., Shimma, S., Taira, S.,

Hatanaka, K., Morone, N., Yao, I., Campbell, P. K., Yuasa, S., Janke, C., Macgregor, G. R., and Setou, M. (2007) Proc Natl Acad Sci U S A 104, 3213-3218

Supplemental Figure Legends Supplemental Fig. 1. Phylogenic tree of TTLL proteins. The 6 ttll proteins from C. elegans were compared with those from H. sapiens and D. melanogaster. Predicted proteins of C. elegans, H. sapiens, and D. melanogaster are colored blue, black, and red, respectively. Circles indicate the clade; 12 clades are shown in this figure. Proteins in the grey and yellow areas are putative tubulin polyglutamylase and polyglycylase, respectively. TTL is the only enzyme that has tubulin tyrosination activity. Note that C. elegans does not have enzymes with both tubulin polyglycylation and tyrosination activities. Supplemental Fig. 2. Immunohistochemical analysis of the head and tail region by using monoclonal antibodies (GT335 and B3) that recognize polyglutamylated tubulins. Signals can be observed in the amphids (Amp), inner labial neurons (white arrows), outer labial neurons (yellow arrows) and some the dendrites (arrowheads), as well as in the PHA, PHB (asterisk), and PQR (double asterisk) neurons. All scale bars = 5 µm. Supplemental Fig. 3. Identification of neurons with a polyglutamylation signal. Double straining with GT335 and anti-GFP polyclonal antibody for neuron-specific GFP. Left column, signal of polyglutamylated tubulin stained by GT335. Middle column, signal of neuron-specific GFP. Right column, merged pictures. Neurons were visualized using the following promoters: ADF, tph-1; ASK, sra-7; AWA, odr-10; AFD, gcy-8 and CEP: dat-1. GFP expressions in Amphid cilia (ADF, ASK, AWA, AFD and ASE) were co-localized with GT335 (top 4 rows and Fig1B, arrowheads), however, that of CEP was not co-localized (bottom row, an arrow). Supplemental Fig. 4. Genomic structures and GFP-fusion genes of 6 ttll genes in C. elegans. Each panel indicates the exon-intron boundary of ttll genes in C. elegans. Red arrows indicate the deletion sites for each gene in the mutants. Green boxes indicate the insertion or fusion point of GFP/mCherry. Supplemental Fig. 5. Expression patterns of ttll genes and transcriptional regulation in sensory neurons. We analyzed the expression and localization pattern of ttll-4. Since ttll-4 has 2 mRNA transcripts—ttll-4a and ttll-4b—that use different promoters (Supplemental Fig. 4), we generated 3 different fusion genes. The ttll-4 transcriptional fusion gene, in which the 1.3-kb promoter of TTLL-4a was fused to GFP (ttll-4a::GFP), shows fluorescence mainly in the body wall and pharyngeal muscles (Supplemental Fig. 5A). Signals were also observed in 1 unidentified sensory neuron in the pharynx (arrowhead in Supplemental Fig. 5A), and weak expression was detected in a few sensory neurons (arrows in Supplemental Fig. 5A). In case of an exon2 fusion gene (ttll-4ab::GFP), signals were additionally detected in many sensory neurons (arrowheads in Supplemental Fig.5B) and motor neurons (Supplemental Fig. 5C), indicating that the specific enhancer of expression in sensory neurons is located in 1st intron of ttll-4a mRNA, which can also work as an alternative promoter for ttll-4b mRNA (Supplemental Fig. 4). On the other hand, the TTLL-4 translational fusion gene was localized in muscles and specifically in the cilia and cell body of sensory neurons (Supplemental Fig. 5D). This localization is consistent with the results that ttll-4 is essential for the polyglutamylation of sensory cilia. Strong signals were not observed in the cilia of both transcriptionally fused ttll-4a::GFP and partially fused ttll-4ab::GFP; this suggests that there is a specific mechanism for transferring TTLL-4 to cilia. (E) ttll-5 is strongly expressed in body wall muscles and not in sensory neurons. (F) TTLL-9 is expressed in entire sensory neurons. (G, H) Both alternative promoters of ttll-11 (ttll-11a and ttll-11b) drive the expression of amphid sensory neurons, however, TTLL-11 protein were localized in axons (an arrow in Supplemental Fig. 6I). (J) TTLL-15 was expressed in many tissues including hypodermis and pharyngeal muscles.

Supplemental Fig. 6. Structural comparison of C.elegans and mouse CCPs. Two C.elegans CCPs (CCPP-1 and CCPP-6) and six mouse CCPs (CCP1-6) are colored yellow and blue, respectively. CCP5-specific insertion is indicated by green bar. Essential residues in CCPs (HxxE sequence, R145, H196, and E270; by convention in the field, these active site residues are numbered based on the mature form of bovine CPA1) are maintained in all CCPs. N-terminal pro-domains (90-100 amino acids) are specifically maintained in all CCPs. CCPP-1 is close to CCP1 (43% amino acid identity) and CCP4 (38%) (Fig. 2A). CCPP-6 is close to CCP5 (26%) and CCP6 (47%) (Fig. 2A). No insertion like CCP5 exists in CCPP-6. Supplemental Fig. 7. Expression pattern of the CCP genes of C. elegans. A, Genome structure and GFP-fusion genes of ccpp-6. Signals of ccpp-6 translationally fused GFP (CCPP-6::GFP) in the head region (B) as well as ccpp-1 transcriptionally fused with GFP (C, ccpp-1::GFP) and ccpp-1 translationally fused with mCherry (D, CCPP-1::mCherry) are shown. Arrowheads indicate the localization of fusion protein in sensory neurons. Scale bars = 5 µm. Supplemental Fig. 8. CCP5 could show deglutamylase activity to some proteins as well as tubulin. The cell lysates from 293T cells transfected with Myc-CCP5 or control vector were analyzed by western blotting using GT335 antibody. Clearly, two bands detected in the control sample completely disappeared (arrowheads) with a tubulin band (arrow). Supplemental Fig. 9. Effect of used buffer and salt concentration on deglutamylase activity of CCP5. In vitro deglutamylation assay were performed using Tris- or PIPES-based buffers in the presence (50 mM or 100 mM) or absence of NaCl. Both Tris and PIPES buffer can functions. On the other hand, the concentration of NaCl strongly influenced the activity. Supplemental Fig. 10. Myc-CCP5 was degraded during immunoprecipitation. CCP5-expressing and control HEK293T cell lysates were immunoprecipitated with anti-Myc polyclonal antibody and analyzed western blotting using anti-Myc monoclonal antibody (9E10). An asterisk indicates Myc-CCP5. Arrows indicates putative degraded fragments of Myc-CCP5. Supplemental Movie 1. Three-dimensional picture of worm head with tubulin polyglutamylation signals in sensory neurons. QuickTime movie was generated by stacking the pictures of different focal plains obtained by confocal microscopy FV1000 using FV10-ASW software (Olympus). Signals are detected in the ciliary structure of amphid, inner labial and outer labial neurons.

ttll genes Sequence names Genetic Positions Expressions Alleles Mutant phenotypes Notesttll-4 ZK1128.6 III: 1.87 Sensory neurons,

Motor neurons,Body wall muscles

tm3310 Complete loss of tubulinpolyglutamylation in ciliaryneurons,IFT defects1,chemosensory defects1.

Expression in sensory cilia isregulated by the enhancer in1st exon.

ttll-5 C55A6.2 V: 3.17 Body wall muslces tm3360 Polyglutamylation signal insensory did not changed.

ttll-9 F25C8.5 V. 25.76 Sensory neuronsIntestine

tm3889 Slight reduction of tubulinpolyglutamylation inciliary neurons,

ttll-11 H23L24.3 IV: 3.94 Sensory neurons,Body wall musclesIntestine

gk482tm4059

gk482:Not identifiedtm4059: No data.

gk482: only ttll-11b-specificexon is deleted.tm4059: common exons of bothttll-11a and ttll-11b are deleted.

ttll-12 D2013.9 II: 1.29 Intestine,Rectal gland cells

not available Unknown.

ttll-15 K07C5.7 V. 2.51 Widely expressed inmany tissues suchas body wallmuscles andpharynx.

tm3871 Polyglutamylation signal did notchanged.

Putative ortholog of notidentified inmammalian genome but seenin ciliates.

Supplemental Table 1 Summary of ttll gene of C.elegans

1 Kimura, Kaplan, Iino, Kunitomo, Blacque, and Setou (unpublished observation)