Phosphatidylinositol 4 phosphate [PI(4)P] is essential for secretion in yeast, but its role in mammalian cells is unclear. Current paradigms propose that PI(4)P acts primarily as a precursor to phosphatidylinositol 4,5 bisphosphate (PIP2), an important plasma membrane regulator. We found that PI(4)P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase, to determine whether PI(4)P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI(4)P, blocks the recruitment of clathrin adaptor AP-1 complexes to the Golgi, and inhibits AP-1-dependent functions. This AP-1 binding defect is rescued by adding back PI(4)P. In addition, purified AP-1 binds PI(4)P, and anti-PI(4)P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI(4)P-rich domains that specify the docking of the AP-1 coat machinery.

Membrane fusion is believed to proceed via intermediate structures called stalks. Mathematical analysis of the stalk provided the elastic energy involved in this structure and predicted the possible evolution of the overall process, but the energies predicted by the original model were suspiciously high. This was due to an erroneous assumption, i.e., that the stalk has a figure of revolution of a circular arc. Here we abandon this assumption and calculate the correct shape of the stalk. We find that it can be made completely stress free and, hence, its energy, instead of being positive and high can become negative, thus facilitating the fusion process. Based on our new calculations, the energies of hemifusion, of complete fusion, and of the pore in a bilayer were analyzed. Implications for membrane fusion and lipid phase transitions are discussed.

The Atg8 autophagy proteins are essential for autophagosome biogenesis and maturation. The γ-aminobutyric acid receptor-associated protein (GABARAP) Atg8 family is much less understood than the LC3 Atg8 family, and the relationship between the GABARAPs' previously identified roles as modulators of transmembrane protein trafficking and autophagy is not known. Here we report that GABARAPs recruit palmitoylated PI4KIIα, a lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) and binds GABARAPs, from the perinuclear Golgi region to autophagosomes to generate PI4P in situ. Depletion of either GABARAP or PI4KIIα, or overexpression of a dominant-negative kinase-dead PI4KIIα mutant, decreases autophagy flux by blocking autophagsome:lysosome fusion, resulting in the accumulation of abnormally large autophagosomes. The autophagosome defects are rescued by overexpressing PI4KIIα or by restoring intracellular PI4P through "PI4P shuttling." Importantly, PI4KIIα's role in autophagy is distinct from that of PI4KIIIβ and is independent of subsequent phosphatidylinositol 4,5 biphosphate (PIP 2 ) generation. Thus, GABARAPs recruit PI4KIIα to autophagosomes, and PI4P generation on autophagosomes is critically important for fusion with lysosomes. Our results establish that PI4KIIα and PI4P are essential effectors of the GABARAP interactome's fusion machinery.autophagy | GABARAP | PI4P | PI4KIIα | autophagosome:lysosome fusion

Phosphatidylinositolpolyphosphates (PIPs) are centrally involved in many biological processes, ranging from cell growth and organization of the actin cytoskeleton to endo-and exocytosis. Phosphorylation of phosphatidylinositol at the D-4 position, an essential step in the biosynthesis of PIPs, appears to be catalyzed by two biochemically distinct enzymes. However, only one of these two enzymes has been molecularly characterized. We now describe a novel class of phosphatidylinositol 4-kinases that probably corresponds to the missing element in phosphatidylinositol metabolism. These kinases are highly conserved evolutionarily, but unrelated to previously characterized phosphatidylinositol kinases, and thus represent the founding members of a new family. The novel phosphatidylinositol 4-kinases, which are widely expressed in cells, only phosphorylate phosphatidylinositol, are potently inhibited by adenosine, but are insensitive to wortmannin or phenylarsine oxide. Although they lack an obvious transmembrane domain, they are strongly attached to membranes by palmitoylation. Our data suggest that independent pathways for phosphatidylinositol 4-phosphate synthesis emerged during evolution, possibly to allow tight temporal and spatial control over the production of this key signaling molecule.Phosphatidylinositol 4-kinases (PtdIns 1 4-kinases) catalyze the phosphorylation of phosphatidylinositol (PtdIns) on the D-4 position of the inositol ring. The product of this reaction, phosphatidylinositol 4-phosphate (PtdIns4P) is a major precursor in the synthesis of phosphatidylinositolpolyphosphates (PIPs), including PtdIns3,4P 2 , PtdIns4,5P 2 , and PtdIns3,4,5P 3 , which participate in signal transduction, membrane trafficking, and cytoskeletal reorganization (1-5). Two classes of PtdIns 4-kinases, Types II and III, have been identified; the enzyme originally designated Type I was subsequently found to be a PtdIns 3-kinase (6). Mammalian Type II PtdIns 4-kinase (henceforth referred to as PI4KII) is a 55-kDa integral membrane protein believed to account for most of the PtdIns 4-kinase activity in cells (7). It can be distinguished from the Type III kinases (PI4KIIIs) by virtue of its lower K m values for ATP and PtdIns, its insensitivity to inhibition by wortmannin, and its sensitivity to adenosine and monoclonal antibody 4C5G (8). The two closely related Type III kinases, III␣ and III␤, belong to the PtdIns 3/4-kinase superfamily, which also includes the protein kinases TOR/ATM/DNA-PK (9, 10). Although PI4KII was identified more than 30 years ago (11) and purified to apparent homogeneity more than 10 years ago (12-16), it had, until now, not been possible to clone it. Here we report the amino acid sequence of rat PI4KII and show that it represents a large family of putative lipid kinases highly conserved from yeast to humans, but bearing little similarity to other known lipid or protein kinases. Recombinant rat PI4KII has the enzymatic characteristics expected of the Type II kinases, and, despite lacking an obvious transmem...

Abstract. Myosin I, a nonfilamentous single-headed actin-activated ATPase, has recently been purified from mammalian tissue (Barylko, B., M. C. Wagner, O. Reizes, and J. E Albanesi. 1992. Proc. Natl. Acad. Sci. . To investigate the distribution of this enzyme in cells and tissues mAbs were generated against myosin I purified from bovine adrenal gland. Eight antibodies were characterized, five of them (M4-M8) recognize epitope(s) on the catalytic "head" portion of myosin I while the other three (M1-M3) react with the "tail" domain.Immunoblot analysis using antiadrenal myosin I antibody M2 demonstrates the widespread distribution of the enzyme in mammalian tissues. Myosin I was immunolocalized in several cell types including bovine kidney (MDBK), rat kidney (NRK), rat brain, rat phaeochromocytoma (PC12), fibroblast (Swiss 3T3), and CHO cells. In all cases, myosin I was concentrated at the cell periphery. The most intense labeling was observed in regions of the cell usually associated with motile activity (i.e., filopodia, lamellipodia and growth cones). These results are consistent with earlier observations on protozoan myosin I that suggest a motile role for the enzyme at the plasma membrane.