Assessing Extrinsic Membrane Protein Dependency to PI4P Using a Plasma Membrane to Endosome Relocalization Transient Assay in Nicotiana benthamiana

International audiencePhosphoinositides are key players from which the various membranes of the cells acquire their identity. The relative accumulation of these low-abundant anionic phospholipids in the cytosolic leaflet of the plasma membrane and of various organelles generates a landmark code, responsible for the selective recruitment of extrinsic proteins at given membranes. One of the key players in the protein/lipid interaction at the plasma membrane in plant cells, is phosphatidylinositol 4-phosphate (PI4P), which patterns the recruitment of effector proteins from the plasma membrane to organelles along the endocytic pathway. Here we describe a fast assay to assess the requirement of PI4P for membrane localization of extrinsic membrane proteins in vivo. This system relies on perturbing PI4P distribution in plant cells via the action of a PI4P phosphatase that depletes the pool of PI4P at a given membrane. This system efficiently decreases PI4P levels, and can therefore be easily used to assess requirement of PI4P (and electrostatics) for the targeting of extrinsic membrane proteins to the plasma membrane or endosomes. Ultimately, this system could also be extended to test the phosphatase activity in planta of enzymes putatively involved in PI4P metabolism

Intracellular phosphatidylserine is essential for retrograde membrane traffic through endosomes

Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides

Microtubules induce self-organization of polarized PAR domains in Caenorhabditis elegans zygotes

A hallmark of polarized cells is the segregation of the PAR polarity regulators into asymmetric domains at the cell cortex(1, 2). Antagonistic interactions involving two conserved kinases, atypical protein kinase C (aPKC) and PAR-1, have been implicated in polarity maintenance(1, 2), but the mechanisms that initiate the formation of asymmetric PAR domains are not understood. Here, we describe one pathway used by the sperm-donated centrosome to polarize the PAR proteins in Caenorhabditis elegans zygotes. Before polarization, cortical aPKC excludes PAR-1 kinase and its binding partner PAR-2 by phosphorylation. During symmetry breaking, microtubules nucleated by the centrosome locally protect PAR-2 from phosphorylation by aPKC, allowing PAR-2 and PAR-1 to access the cortex nearest the centrosome. Cortical PAR-1 phosphorylates PAR-3, causing the PAR-3/aPKC complex to leave the cortex. Our findings illustrate how microtubules, independent of actin dynamics, stimulate the self-organization of PAR proteins by providing local protection against a global barrier imposed by aPKC