C. elegans methods, strains and nomeclature
The wild type Bristol N2 strain was used for all experiments and was handled under standard laboratory conditions as described previously . Standard methods were used for DNA microinjection  and dsRNA synthesis and microinjection . Nomenclature is in agreement with available Genetic Nomenclature for Caenorhabditis elegans  and has been approved prior to manuscript submission.
Wormbase (2002–2004 versions and freeze versions [15, 38, 39]) databases were repeatedly searched for human α-GAL and α-NAGA orthologs using the BLASTP  program set at default values. Amino acid sequences of human lysosomal α-GAL and α-NAGA (acc. no. NP_000160 and acc. no. NP_000253 ) were used as query sequences.
cDNA amplification and sequencing
Total RNA was isolated from mixed stages of N2 cultures  and reverse transcribed with an oligodT-T7 (5'-AATACGACTCACTATAG) primer and Superscript reverse transcriptase (Invitrogen). The entire coding region of R07B7.11 was PCR amplified in two overlapping PCR products, with intragenic primers designed according to available Wormbase  and Worfdb  data. SL1 primer (5'GGTTTAATTACCCAAGTTTGAG) and SL2 primer (5'GGTTTTAACCCAGTTACTCAAG)  together with gene specific primer (5'ATCCTGATTAATTTTAATTGC) were used to amplify 942 bp of the 5' part of the cDNA and to evaluate the mode of trans splicing; the 1142 bp fragment of the 3' end of cDNA was amplified with T7 primer and a gene specific primer (5'CTTAAGTTTGGAATTTATGAA). The dominant PCR products were cloned with TOPO TA cloning kit (Invitrogen) into the pCR 2.1 vector. Positive clones were sequenced using the Li-Cor automated fluorescent sequencer and sequences were aligned with R07B7 reference cosmid sequence in the AlignIR software (Li-Cor) to evaluate splicing boundaries and overall gene organization.
Multiple alignment and phylogenetic analyses
Confirmed or predicted amino acid sequences of melibiase family members  representing plant, unicellular, and animal kingdoms were aligned using ClustalW algorithm  and Blosum62 matrix. The SwissProt/TrEMBL  accesion code and source organism of the sequences are depicted in Figure 2. The sequence alignment was used for phylogenetic analysis with the software package PHYLIP . The phylogenetic tree is based on 100 bootstraped input alignments and was constructed by maximum likelihood method with Jones-Taylor-Thornton matrix model . Sequence identities between species were calculated without signal sequence in EMBOSS by Needleman-Wunsch global alignment algorithm with Blosum62 matrix, gap penalty – 10 and gap extension penalty – 0.5 [8, 48, 49]. Signal peptides were predicted at the SignalP server  both by algorithms using neural networks and Hidden Markov Models. The results were compared to known signal sequences. The differences between signal peptides predicted by the algorithms are depicted in Figure 2.
The 3D model of GANA-1 is based on the X-ray structure of chicken α-NAGA, rice α-GAL and human α-GAL (PDB codes 1ktcA, 1uas and 1r47, respectively) [7–9, 51]. The model was created using the automated homology modeling server SwissModel with structure refinement and model evaluation in the DeepView program . The print quality figures (Figure 4) and animations (Additional file 1) were ray traced using PovRay software package .
Transgenic GFP expression
The entire coding region of the gana-1 gene, including 3 kb of its 5'upstream sequence, was amplified from N2 genomic DNA through a nested PCR reaction using DyNAzyme EXT™ PCR system (Finnzymes) and two pairs of primers: the external pair (5'GTGAGAGTGGGGAGATAGAA and 5'TCAATTTGCTTGAGGTACATA) and the internal primers, with overhangs containing SphI and SalI restriction sites respectively (5'ACATGCATGCAACTTTCACAGGAACACAAC and 5'CGACGTCGACAATTGAACTCTATTGGTTCTCAA). The amplified DNA fragment (4709 bp) was cloned using TOPO-XL cloning kit (Invitrogen) into the pCR-XL-TOPO vector. The SphI and SalI gana-1 restriction fragment was recloned into the GFP reporter vector pPD95.67 (supplied by A. Fire, Stanford University). The in-frame nature of the insert was confirmed by sequencing. The green fluorescent protein (GFP) fusion construct pJH3 (50 ng/μl) and pRF4 plasmid (50 ng/μl) used as the dominant marker were co-injected into the gonads of young adult N2 worms. Transgenic animals were screened for GFP signal. Nikon Eclipse E800 with C1 confocal module and 488 nm and 543 nm lasers and differential interference contrast (DIC) optics was used for specimen examination. EZ-C1 software (Nikon) was used for picture analysis and 3D rendering (Additional Files 2, 3).
Alkalization of acidic cell compartment
Mixed stage pJH3 and N2 (control) cultures were harvested from NGM OP50 plates and washed with water. Worms were pelleted by centrifugation (max. 1000 RPM, 2 min.) between the washes. Worms were treated with either one of two agents (NH4Cl, concanamycin A – CON A) [33, 34], that are known to specifically increase pH in the cellular acidic compartment. For the NH4Cl method, animals were suspended in 0, 10, 25, 50, 75 and 100 mM aqueous solutions of NH4Cl. Small aliquots of worms were examined after 30 min, 2, 4, 6, 8 and 24 hours. For CON A, animals were suspended in 0, 10, 20, 50, 100, 150, 200 nM solutions of CON A in aqueous media. Small aliquots of worms were examined after 1, 3, 6 and 24 hours.
Microscopical examination was performed as described above.
The fixation and immunofluorescence staining procedures were based on the approaches of Nonet et al. . Mixed stage N2 cultures were harvested from NGM OP50 plates and washed thoroughly in M9 buffer to remove intestinal bacteria. Worms were pelleted by centrifugation (1000 RPM, 2 min.) between the washes. Worms were fixed overnight in 4% paraformaldehyde in 100 mM sodium/potassium phosphate buffer. Afterwards the pellets were washed three times in 1 × PBS, and incubated in 1% Triton X-100, 100 mM Tris (pH 7.0), 1% β-Mercaptoethanol overnight at 37°C to reduce the cuticle. After 5 washes in 1 × PBS, the worms were incubated for 5 hours in 900 U/ml collagenase type IV (Sigma) diluted in Krebs-Ringer solution (119 mM NaCl, 25 mM NaHCO3, 11.1 mM glucose, 1.6 mM CaCl2·H2O, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4·7H2O, pH 7.4). The reduction/digestion step was performed twice. Pellets were washed 3 times with 1 × PBS and stored for further processing in AbA buffer (1 × PBS, 0.1% Triton X-100, 1% BSA, 0.05% NaN3). AbA buffer was used for antibody dilution. Primary antibody (polyclonal rabbit anti-GFP IgG (Abcam)) was diluted 1:500. Secondary antibody (goat anti-rabbit IgG Alexa Fluor 488 labeled (Molecular Probes)) was diluted 1:1000. Both incubations were performed overnight at room temperature, with AbB buffer (1 × PBS, 0.1% Triton X-100, 0.1% BSA, 0.05% NaN3) washes in between.
Nuclei were counterstained with SYTOX orange (Molecular Probes) and the microscopic evaluation was performed as described above.
Mixed stage pJH3 and N2 cultures were harvested from NGM OP50 plates. Worms were homogenized by sonication and the concentration of protein was measured by the Hartree method . The proteins (equivalent of 25–50 μg of protein per lane) were separated by SDS-PAGE gradient gel (4% to 20% polyacrylamide) and transferred onto nitrocellulose membrane by semi-dry blotting. The membrane was treated according to a common Western blotting protocol with chemiluminiscence detection (SuperSignal, West Pico) . Rabbit polyclonal anti-GFP IgG (Abcam, dilution 1:5000) was used as the primary antibody, the secondary antibody was goat anti-rabbit IgG/Px (Pierce, diluted 1:20 000).
RNA mediated interference
The PCR product containing the entire gana-1 cDNA was cloned into pCRII-TOPO vector (Invitrogen) and L4440 double promoter vector for microinjection and feeding RNAi respectively. In-vitro transcription employing T7 and SP6 RNA polymerases (Promega) was used to generate antisense single stranded RNA molecules, which were annealed to generate double stranded RNA (dsRNA). dsRNA was microinjected into N2 worms which were fed on HT115 E. coli strain carrying L4440 plasmid with gana-1 insert. The F1 and early F2 progeny was screened for morphological phenotypes. N2 worms microinjected with water and fed on HT115 E. coli transformed with L4440 vector without insert were used as a control. 5–7 worms were microinjected both with dsRNA and water in each of 4 separate experiments, single worm progeny reaching 110–150 individuals.
Determination of α-GAL and α-NAGA and β-hexosaminidase activities
Prior to all activity measurements worms were washed from culture plates and repeatedly (6 times) washed and centrifuged in M9 buffer and finally pelleted by centrifugation. 4-methylumbelliferyl (MU)-α-D-N-acetylgalactosaminide (1 mM), 4-MU-α-D-galactopyranoside and 4-MU-β-D-glucopyranoside in theMcIlvaine buffer (0.1 M citrate/0.2 M phosphate buffer at acidic pH) were used as enzyme substrates. Reaction mixtures (sample and enzyme substrate) were incubated at 37°C and reactions were stopped by 600 μl of 0.2 M glycine/ NaOH buffer (pH 10.6) [13, 57]. Fluorescence signal of the 4-methylumbelliferone was measured on the luminiscence spectrofotometer LS 50B (Perkin Elmer) (emission 365 nm and excitation 448 nm). Inhibitors (N-acetyl-D-galactosamine, D-galactose and D-glucose) were used in 0.1 M final concentration. All measurements were performed in doublets.