Abstract:Phosphoinositides are lipids involved in the vesicular transport of proteins and lipids between the different compartments of eukaryotic cells. They act by recruiting and/or activating effector proteins and thus are involved in regulating various cellular functions, such as vesicular budding, membrane fusion and cytoskeleton dynamics. Although detected in small concentrations in membranes, their role is essential to cell function, since imbalance in their concentrations is a hallmark of many cancers. Their synthesis involves phosphorylating/dephosphorylating positions D3, D4 and/or D5 of their inositol ring by specific lipid kinases and phosphatases. This process is tightly regulated and specific to the different intracellular membranes. Most enzymes involved in phosphoinositide synthesis are conserved between yeast and human, and their loss of function leads to severe diseases (cancer, myopathy, neuropathy and ciliopathy).Keywords: lipids; membrane trafficking; vesicles; phosphoinositides; phosphatase; kinase Phosphoinositides in Cellular Membranes Lipids, Major Membrane ComponentsThe dynamic modulation of the physicochemical properties of membranes is required for eukaryotic cells function. Indeed, cells live in an environment characterized by temperature, relative humidity, pH, sun exposure, osmotic pressure and nutrient variations. Living organisms have to adapt to variations of these different factors in order to keep their intracellular balance. Eukaryotic cells have achieved this by adopting a compartmentalized organization, which minimizes the intracellular variations resulting from extracellular fluctuations. The plasma membrane is the first barrier separating the cytoplasm from the extracellular medium. Its composition ensures a mechanical protection, but also allows exchanges with the medium through transporters and receptors, as a form of very selective permeability.Membranes are composed of two phospholipid leaflets organized as a bilayer in which sterols, glycolipids and proteins are inserted. The phospholipids of this bilayer are amphiphilic with a hydrophilic group (head) linked to a hydrophobic group (tail) ( Figure 1A). In the bilayer, the hydrophobic groups face each other, thus creating a hydrophobic space in between them, which ensures its role as a barrier. This property is very important for the anchoring of hydrophobic molecules, such as sterols or ceramides, transmembrane domains or the lipid anchor of proteins. The lipid composition of membranes varies according to the organism (eukaryotes or prokaryotes), the cell type (among the different tissues of a multicellular organism), the membrane type (plasma Int.

The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic models. The secretory pathway was the first trafficking pathway clearly understood mainly thanks to the work done in the laboratory of Randy Schekman in the 1980s. They have isolated yeast sec mutants unable to secrete an extracellular enzyme and these SEC genes were identified as encoding key effectors of the secretory machinery. For this work, the 2013 Nobel Prize in Physiology and Medicine has been awarded to Randy Schekman; the prize is shared with James Rothman and Thomas Südhof. Here, we present the different trafficking pathways of yeast S. cerevisiae. At the Golgi apparatus newly synthesized proteins are sorted between those transported to the plasma membrane (PM), or the external medium, via the exocytosis or secretory pathway (SEC), and those targeted to the vacuole either through endosomes (vacuolar protein sorting or VPS pathway) or directly (alkaline phosphatase or ALP pathway). Plasma membrane proteins can be internalized by endocytosis (END) and transported to endosomes where they are sorted between those targeted for vacuolar degradation and those redirected to the Golgi (recycling or RCY pathway). Studies in yeast S. cerevisiae allowed the identification of most of the known effectors, protein complexes, and trafficking pathways in eukaryotic cells, and most of them are conserved among eukaryotes.

Myotubularin MTM1 is a phosphoinositide (PPIn) 3-phosphatase mutated in X-linked centronuclear myopathy (XLCNM; myotubular myopathy). We investigated the involvement of MTM1 enzymatic activity on XLCNM phenotypes. Exogenous expression of human MTM1 in yeast resulted in vacuolar enlargement, as a consequence of its phosphatase activity. Expression of mutants from patients with different clinical progression and determination of PtdIns3P and PtdIns5P cellular levels confirmed the link between vacuolar morphology and MTM1 phosphatase activity, and showed that some disease mutants retain phosphatase activity. Viral gene transfer of phosphatase-dead myotubularin mutants (MTM1C375S and MTM1S376N) significantly improved most histological signs of XLCNM displayed by a Mtm1-null mouse, at similar levels as wild-type MTM1. Moreover, the MTM1C375S mutant improved muscle performance and restored the localization of nuclei, triad alignment, and the desmin intermediate filament network, while it did not normalize PtdIns3P levels, supporting phosphatase-independent roles of MTM1 in maintaining normal muscle performance and organelle positioning in skeletal muscle. Among the different XLCNM signs investigated, we identified only triad shape and fiber size distribution as being partially dependent on MTM1 phosphatase activity. In conclusion, this work uncovers MTM1 roles in the structural organization of muscle fibers that are independent of its enzymatic activity. This underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity.