Supplemental Material

This article contains the following supporting material:

• Figure 1 - Coding nucleotide sequence and protein sequence for ppk-3. Predicted intron number six from VF11C1L.1 (http://www.wormbase.org/) is found in cDNA clones, and the N-terminal of the protein sequence was extended based on protein conservation with other species and on the fact that a possible FYVE domain is entirely present at the N-terminus (not the case in the WormBase VF11C1L.1 sequence). At the top is depicted the predicted cDNA sequence, below is the deduced amino acid sequence. Both are numbered on the left. This newly predicted ppk-3 gene sequence encompasses 14 exons (exon-exon boundaries are indicated by **) and encodes a 1497 amino acids protein, whose protein domains are underlined and indicated as follow: a FYVE (Fab1p, YOTB, Vac1p, EEA1) domain from amino acids 102 to 171, a Cpn60/TCP-1 chaperonin family domain from amino acids 297 to 690, and a lipid kinase domain from amino acids 1194 to 1482. The nucleotide binding motif of the kinase domain is located at amino acids 1263 to 1280, and the catalytic loop and magnesium coordination residues are located at amino acids 1352 to 1356. Point mutations in ppk-3 mutants are S1356F (n2668) and Q1487X (n2835).

• Figure 2 - ppk-3 expression. Wild-type worms injected with ppk-3::YFP fusion. (A) Projection from confocal sections of a L1 larva. The construct is expressed in some neurons of the head and tail, some neurons of the ventral (large arrow) and dorsal (small arrow) nerve cords and some commissures (arrow head). (B) Fluorescent microscopy picture of a L1 larva showing a mosaic intestinal expression of ppk-3. (C) Confocal section of neurons of the ventral nerve cord of an adult worm showing punctuate cytoplasmic localization of ppk-3. Scale bar=5 μm

• Figure 3 - C.elegans RAB-7 specificity towards late endosomes and lysosomes. (A) Confocal pictures of wild-type and ppk-3 (n2668) mutant coelomocytes expressing RAB-7::GFP. Scale bar=5 μm. (B) Confocal pictures of coelomocytes expressing RME-8::GFP or RAB-7::GFP 24 hours after the injection of the fluid-phase marker Texas-Red BSA (red). After 24 hours, Texas-Red BSA has been internalized into lysosomes (see figure 6B). RME-8 positive late endosomes are not filled with Texas-Red BSA, whereas some RAB-7 positive vesicles are filled with Texas-Red BSA. Scale bar=5 μm.

• Figure 4 - ppk-3 and cup-5 genetic interaction (A) Lethality and maternal effect of ppk-3(n2668), cup-5(ar465) single mutants and of cup-5(ar465); ppk-3(n2668) double mutant. (B) Confocal pictures of wild-type and ppk-3(n2668) coelomocytes expressing CUP-5::GFP. Enlarged vacuoles of ppk-3 mutant are indicated by stars. Scale bar=5 μm.