The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.

SummaryAutophagy is a conserved degradative transport pathway. It is characterized by the formation of double-membrane autophagosomes at the phagophore assembly site (PAS). Atg18 is essential for autophagy but also for vacuole homeostasis and probably endosomal functions. This protein is basically a b-propeller, formed by seven WD40 repeats, that contains a conserved FRRG motif that binds to phosphoinositides and promotes Atg18 recruitment to the PAS, endosomes and vacuoles. However, it is unknown how Atg18 association with these organelles is regulated, as the phosphoinositides bound by this protein are present on the surface of all of them. We have investigated Atg18 recruitment to the PAS and found that Atg18 binds to Atg2 through a specific stretch of amino acids in the b-propeller on the opposite surface to the FRRG motif. As in the absence of the FRRG sequence, the inability of Atg18 to interact with Atg2 impairs its association with the PAS, causing an autophagy block. Our data provide a model whereby the Atg18 b-propeller provides organelle specificity by binding to two determinants on the target membrane.

The retinoblastoma protein (pRB) is a tumor suppressor and key regulator of the cell cycle. We have previously shown that pRB interacts with phosphatidylinositol-4-phosphate 5-kinases, lipid kinases that can regulate phosphatidylinositol 4,5-bisphosphate levels in the nucleus. Here, we investigated pRB binding to another lipid kinase in the phosphoinositide cycle, diacylglycerol kinase (DGK) that phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. We found that DGK, but not DGK␣ or DGK, interacts with pRB in vitro and in vivo. Binding of DGK to pRB is dependent on the phosphorylation status of pRB, since only hypophosphorylated pRB interacts with DGK. DGK also binds to the pRB-related pocket proteins p107 and p130 in vitro and in cells. Although DGK did not affect the ability of pRB to regulate E2F-mediated transcription, we found that pRB, p107, and p130 potently stimulate DGK activity in vitro. Finally, overexpression of DGK in pRB-null fibroblasts reconstitutes a cell cycle arrest induced by ␥-irradiation. These results suggest that DGK may act in vivo as a downstream effector of pRB to regulate nuclear levels of diacylglycerol and phosphatidic acid. Diacylglycerol (DAG)3 regulates many cellular processes, including proliferation, differentiation, and cell migration, by modulating the activity of several proteins, such as protein kinase C (PKC), Ras guanyl nucleotidereleasing proteins, chimaerins, and Munc 13 (1). DAG can be produced by the action of several different signal transduction pathways, including phospholipase C-mediated hydrolysis of phosphoinositides or phosphatidylcholine and phospholipase D-mediated hydrolysis of phosphatidylcholine followed by dephosphorylation of phosphatidic acid (PA), and during de novo synthesis of phospholipids (2).DAG is not only produced at the plasma membrane but at other intracellular sites as well, including the nucleus. Nuclear DAG levels are increased in liver as a consequence of two-thirds partial hepatectomy (3) and in cell cultures treated with insulin-like growth factor 1, which stimulates proliferation (4, 5). This suggests that nuclear DAG levels are intimately linked with cell cycle progression, but a causal relationship has not been firmly established. An attractive hypothesis is that nuclear DAG stimulates cell cycle progression via a DAG-binding protein such as PKC (1, 6). Indeed, DAG in the nucleus recruits and activates PKC in response to insulin-like growth factor 1 stimulation of Swiss 3T3 cells, which is required for G 1 to S phase transition (4, 7). However, the role of PKC in regulating the cell cycle is complex, with different PKC isoforms inducing a cell cycle arrest or stimulation of cell cycle progression. Furthermore, the same PKC isoform is able to induce both an arrest and progression through the cell cycle when expressed in different cell types (8).In the nucleus, DAG kinase (DGK) controls the levels of DAG generated from PI-phospholipase C-mediated hydrolysis of PI(4,5)P 2 (9), and nuclear DGK activity can be sti...