SUMMARYType II phosphatidylinositol 4-kinase (PI4KII) produces the lipid phosphatidylinositol 4-phosphate (PI4P), a key regulator of membrane trafficking. Here, we generated genetic models of the sole Drosophila melanogaster PI4KII gene. A specific requirement for PI4KII emerged in larval salivary glands. In PI4KII mutants, mucin-containing glue granules failed to reach normal size, with glue protein aberrantly accumulating in enlarged Rab7-positive late endosomes. Presence of PI4KII at the Golgi and on dynamic tubular endosomes indicated two distinct foci for its function. First, consistent with the established role of PI4P in the Golgi, PI4KII is required for sorting of glue granule cargo and the granule-associated SNARE Snap24. Second, PI4KII also has an unforeseen function in late endosomes, where it is required for normal retromer dynamics and for formation of tubular endosomes that are likely to be involved in retrieving Snap24 and Lysosomal enzyme receptor protein (Lerp) from late endosomes to the trans-Golgi network. Our genetic analysis of PI4KII in flies thus reveals a novel role for PI4KII in regulating the fidelity of granule protein trafficking in secretory tissues.

Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi–derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.

No abstract

Background:The low density lipoprotein receptor-related protein 1 (LRP1) is a transforming growth factor ␤ (TGF-␤) receptor in ovarian cells. Results: GULP is an adapter to LRP1 and mediates TGF-␤ signaling in signaling-competent early endosomes. Conclusion: GULP positively regulates TGF-␤ signaling in ovarian cells. Significance: GULP is poorly expressed in ovarian cancer cells and is a target for TGF-␤-mediated growth inhibition.

T he low-density lipoprotein (LDL) receptor pathway plays a major role in the regulation of the plasma level of LDL in humans. When clearance of LDL particles through the LDL receptor pathway is markedly impaired or abolished, as in familial hypercholesterolemia, 1 profound elevations in plasma LDL result. At the other extreme, more effective clearance of LDL particles by the LDL receptor pathway is the major mechanism by which statins lower plasma LDL. However, these changes in the activity of the LDL receptor pathway are produced by disease or pharmacological intervention and are not evidence of its physiological role.The LDL receptor pathway paradigm stipulates that intracellular cholesterol homeostasis is based on a reciprocal relationship between the rate at which cholesterol within an LDL particle enters the cell through the LDL receptor pathway and the rate at which cholesterol and LDL receptors are synthesized within the cell.2 In brief, cholesterol that enters the cell within an LDL particle is released from the particle in the lysosome and equilibrates with the cholesterol in the endoplasmic reticulum (ER) membrane. The cholesterol mass within the ER membrane determines the activity of the sterol regulatory element-binding protein 2 (SREBP2) pathway, which regulates the synthesis of cholesterol and the LDL receptor.3-5 Additional, more rapid, adaptation mechanisms exist, including esterification of cholesterol by acyl CoA:cholesterol acyltransferase (ACAT) and acute inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCoA reductase, the rate-limiting step of endogenous cholesterol biosynthesis) by hydroxysterols. 6,7 The regulatory paradigm of the LDL receptor pathway is a simple, closed-loop, seesaw homeostatic model: increased uptake is followed by decreased synthesis; decreased synthesis is followed by increased uptake. However, this paradigm was based on studies in cultured fibroblasts, a cell that plays no important role in total body cholesterol homeostasis and a cell with little capacity to secrete cholesterol. In contrast, © 2013 American Heart Association, Inc. Objective-The hypothesis that cholesterol that enters the cell within low-density lipoprotein (LDL) particles rapidly equilibrates with the regulatory pool of intracellular cholesterol and maintains cholesterol homeostasis by reducing cholesterol and LDL receptor synthesis was validated in the fibroblast but not in the hepatocyte. Accordingly, the present studies were designed to compare the effects of cholesterol that enters the hepatocyte within an LDL particle with those of cholesterol that enters via other lipoprotein particles. Approach and Results-We measured cholesterol synthesis and esterification in hamster hepatocytes treated with LDL and other lipoprotein particles, including chylomicron remnants and VLDL. Endogenous cholesterol synthesis was not significantly reduced by uptake of LDL, but cholesterol esterification (280%) and acyl CoA:cholesterol acyltransferase 2 expression (870%) were increased. In contrast, ...

Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.

Secretory granules (SGs) are organelles responsible for regulated exocytosis of biologically active molecules in professional secretory cells. Maturation of SGs is a crucial process in which cargoes of SGs are processed and activated, allowing them to exert their function upon secretion. Nonetheless, the intracellular trafficking pathways required for SG maturation are not well defined. We recently performed an RNA interference (RNAi) screen in Drosophila larval salivary glands to identify trafficking components needed for SG maturation. From the screen, we identified several Rab GTPases (Rabs) that affect SG maturation. Expression of constitutively active (CA) and dominant-negative (DN) forms narrowed down the Rabs important for this process to Rab5, Rab9 and Rab11. However, none of these Rabs localizes to the limiting membrane of SGs. In contrast, examination of endogenously YFP-tagged Rabs (YRabs) in larval salivary glands revealed that YRab1 and YRab6 localize to the limiting membrane of immature SGs (iSGs) and SGs. These findings provide new insights into how Rab GTPases contribute to the process of SG maturation.

No abstract