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

KA1-targeted regulatory domain mutations activate Chk1 in the absence of DNA damage

The Chk1 protein kinase is activated in response to DNA damage through ATR-mediated phosphorylation at multiple serine-glutamine (SQ) residues within the C-terminal regulatory domain, however the molecular mechanism is not understood. Modelling indicates a high probability that this region of Chk1 contains a kinase-associated 1 (KA1) domain, a small, compact protein fold found in multiple protein kinases including SOS2, AMPK and MARK3. We introduced mutations into Chk1 designed to disrupt specific structural elements of the predicted KA1 domain. Remarkably, six of seven Chk1 KA1 mutants exhibit constitutive biological activity (Chk1-CA) in the absence of DNA damage, profoundly arresting cells in G2 phase of the cell cycle. Cell cycle arrest induced by selected Chk1-CA mutants depends on kinase catalytic activity, which is increased several-fold compared to wild-type, however phosphorylation of the key ATR regulatory site serine 345 (S345) is not required. Thus, mutations targeting the putative Chk1 KA1 domain confer constitutive biological activity by circumventing the need for ATR-mediated positive regulatory phosphorylation