In eucaryotic cells, phosphatidylinositol can be phosphorylated on the inositol ring into 7 distinct phosphoinositides (PPIn) which have been implicated in a variety of cellular processes, including actin rearrangement, membrane and protein trafficking, cell survival and mitogenesis. The phosphoinositides act as precursors of second messengers or directly recruit signaling proteins to specific membrane domains. Our aim is to study the regulation of the turn-over, the conversion and the biological roles of a subclass of phosphoinositides, PtdIns(3)P, PtdIns(5)P, PtdIns(3,5)P<sub>2</sub>, which are the substrates or products of the kinase PIKFYVE/Fab1p and of the family of myotubularin (MTM) lipid phosphatases conserved from yeast to human. PtdIns(3)P and PtdIns(3,5)P<sub>2</sub> have been implicated at least in endocytosis while the function of PtdIns(5)P is unknown. Human myotubularin 1 (MTM1) and dynamin 2 (DNM2), a protein known to be involved in the formation of endocytic vesicles at the plasma membrane, are mutated in cases of centronuclear myopathies, suggesting that some PPIn and membrane trafficking are implicated in human pathologies. We have identified several mutants for the C.elegans orthologue of PIKFYVE/Fab1p named PPK-3, a kinase which phosphorylates PtdIns(3)P into PtdIns(3,5)P<sub>2</sub>.
ppk-3 mutant displays a striking enlargement of vacuoles positive for the lysosomal marker LMP-1 in different tissues and for the late endosomal marker RAB-7 in the intestine. Membranes of the enlarged lysosomes originate from smaller lysosomes, and functional and genetic analyses show that the terminal maturation of lysosomes is defective. Protein degradation is not affected in this
ppk-3 mutant and is thus uncoupled from membrane retrieval. We measured the level of PtdIns(3,5)P<sub>2</sub> and showed that its production is impaired in this mutant. This work strongly suggests that the main function of PPK-3 is to mediate membrane retrieval from matured lysosomes through regulation of PtdIns(3,5)P<sub>2</sub>. C.elegans
mtm-1 and
dyn-1 are homologous to human
mtm1 and
dnm2 respectively. To characterise the myotubularin/dynamin pathway, we have used
ppk-3 mutant to modify PtdIns(3,5)P<sub>2</sub> level in
mtm-1 and
dyn-1 mutants. Analysis of the single and double mutants and their impact on endocytosis will be presented.