Primary cilia are microtubule-based membrane projections located at the surface of many cells. Defects in primary cilia formation have been implicated in a number of genetic disorders, such as Bardet-Biedl Syndrome and Polycystic Kidney Disease. Recent studies have demonstrated that polarized vesicular transport involving Rab8 and its guanine nucleotide-exchange factor Rabin8 is essential for primary ciliogenesis. Here we report that Rabin8 is a direct downstream effector of Rab11, which functions in membrane trafficking from the trans-Golgi network and recycling endosomes. Rab11, in its GTP-bound form, interacts with Rabin8 and kinetically stimulates the guanine nucleotide-exchange activity of Rabin8 toward Rab8. Rab11 is enriched at the base of the primary cilia and inhibition of Rab11 function by a dominant-negative mutant or RNA interference blocks primary ciliogenesis. Our results suggest that Rab GTPases coordinate with each other in the regulation of vesicular trafficking during primary ciliogenesis.

When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I–mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor–induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery.

Background: Exocytosis at the plasma membrane mediated by Rabin8 is essential for primary ciliogenesis. Results: Rabin8 activation involves the relief of its autoinhibition. Rabin8 interacts with the exocyst component Sec15 upon its activation. Conclusion: The Rab8 guanine nucleotide exchange factor-effector interaction is important for exocytosis and primary ciliogenesis. Significance: The study sheds light on our understanding of the regulation of exocytosis and ciliogenesis.

The integral membrane lipid phosphatase Sac1p regulates local pools of phosphatidylinositol-4-phosphate (PtdIns(4)P) at endoplasmic reticulum (ER) and Golgi membranes. PtdIns(4)P is important for Golgi trafficking, yet the significance of PtdIns(4)P for ER function is unknown. It also remains unknown how localization of Sac1p to distinct organellar membranes is mediated. Here, we show that a COOH-terminal region in yeast Sac1p is crucial for ER targeting by directly interacting with dolicholphosphate mannose synthase Dpm1p. The interaction with Dpm1p persists during exponential cell division but is rapidly abolished when cell growth slows because of nutrient limitation, causing translocation of Sac1p to Golgi membranes. Cell growth–dependent shuttling of Sac1p between the ER and the Golgi is important for reciprocal control of PtdIns(4)P levels at these organelles. The fraction of Sac1p resident at the ER is also required for efficient dolichol oligosaccharide biosynthesis. Thus, the lipid phosphatase Sac1p may be a key regulator, coordinating the secretory capacity of ER and Golgi membranes in response to growth conditions.

Compartment‐specific control of phosphoinositide lipids is essential for cell function. The Sac1 lipid phosphatase regulates endoplasmic reticulum (ER) and Golgi phosphatidylinositol‐4‐phosphate [PI(4)P] in response to nutrient levels and cell growth stages. During exponential growth, Sac1p interacts with Dpm1p at the ER but shuttles to the Golgi during starvation. Here, we report that a C‐terminal region in Sac1p is required for retention in the perinuclear ER, whereas the N‐terminal domain is responsible for Golgi localization. We also show that starvation‐induced shuttling of Sac1p to the Golgi depends on the coat protein complex II and the Rer1 adaptor protein. Starvation‐induced shuttling of Sac1p to the Golgi specifically eliminates a pool of PI(4)P generated by the lipid kinase Pik1p. In addition, absence of nutrients leads to a rapid dissociation of Pik1p, together with its non‐catalytical subunit Frq1p, from Golgi membranes. Reciprocal rounds of association/dissociation of the Sac1p lipid phosphatase and the Pik1p/Frq1p lipid kinase complex are responsible for growth‐dependent control of Golgi phosphoinositides. Sac1p and Pik1p/Frq1p are therefore elements of a unique machinery that synchronizes ER and Golgi function in response to different growth conditions.

The exocyst is an evolutionarily conserved octameric complex involved in polarized exocytosis from yeast to humans. The Sec3 subunit of the exocyst acts as a spatial landmark for exocytosis through its ability to bind phospholipids and small GTPases. The structure of the N-terminal domain of Sec3 (Sec3N) was determined ab initio and defines a new subclass of pleckstrin homology (PH) domains along with a new family of proteins carrying this domain. Respectively, N-and C-terminal to the PH domain Sec3N presents an additional ␣-helix and two ␤-strands that mediate dimerization through domain swapping. The structure identifies residues responsible for phospholipid binding, which when mutated in cells impair the localization of exocyst components at the plasma membrane and lead to defects in exocytosis. Through its ability to bind the small GTPase Cdc42 and phospholipids, the PH domain of Sec3 functions as a coincidence detector at the plasma membrane.The exocyst is an evolutionarily conserved octameric protein complex composed of subunits Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. This complex was first identified by genetic and biochemical methods in the budding yeast Saccharomyces cerevisiae (1, 2). A homologous complex was subsequently discovered in mammalian cells (3). The exocyst mediates initial tethering of post-Golgi secretory vesicles to the plasma membrane, a step that precedes SNARE 7 -driven membrane fusion (4, 5). The exocyst is regulated by numerous cellular factors, and in particular small GTPases, which are primarily responsible for the spatiotemporal control of exocytosis (6).Recent studies have provided insights into the molecular architecture and function of tethering proteins. Crystal structures of nearly full-length Exo70 (7-9) and large fragments of Sec6 (10), Sec15 (11), and Exo84 (7) have been determined. Despite the lack of sequence similarity, these structures all reveal a similar fold, consisting of elongated tandem repeats of helical bundles, which are predicted to pack against one another during assembly of the exocyst complex (4). The recently determined structure of the yeast Dsl1p complex implicated in Golgi-to-endoplasmic reticulum transport provided the first glance into an assembled tethering complex consisting of helical bundles similar to those of exocyst subunits (12). The structure suggested a similar architecture, and possibly a common origin, among multisubunit tethering complexes. Structures have also been determined of the RalA-binding domains of the mammalian exocyst subunits Sec5 (13) and Exo84 (14), which display immunoglobulin-like and pleckstrin homology (PH) folds, respectively. However, these two domains are missing in the yeast complex and are not considered part of the conserved core of the exocyst (4).Studies in yeast suggest that subunit Sec3 plays a pivotal role in exocyst function and vesicle tethering. Sec3 localizes, together with Exo70, to the growing end of the daughter cell (known as the "bud tip"). Although the localization of other exocyst c...

Pl Sleuth The term "phosphoinositides" describes a class of phosphorylated lipids derived from phosphatidylinositol (PI) and includes the usual suspects: PI(3)P, PI(4)P, and PI(4,5)P2. These molecules are involved in a broad range of intracellular signaling pathways, impacting everything from cell growth and differentiation to cytoskeletal dynamics and membrane trafficking. The discovery of new phosphoinositide binding partners plays an important ongoing role in elucidating the full functionality of this fascinating group of compounds, as well as in identifying targets for the treatment of their related diseases. In this issue, Kndler and Mayinger (p. 858) present a methodology for the identification of novel lipid-binding proteins that improves upon previously used techniques, which had a tendency to suffer from high levels of false positives and assay conditions that could not closely emulate true in vivo conditions. The new assay builds upon previous technologies but overcomes many of the hurdles by making use of artificial liposomes, the content of which can be controlled and varied as required to mirror component concentrations found in vivo. Importantly, this system allows for the use of crude extracts of subcellular fractions without the need for time-consuming purification steps, as demonstrated by the authors using yeast membrane extracts. The continued evolution and improvement of such assays will undoubtedly lead to more rapid advances in our understanding of phosphoinositide signaling and the subsequent development of treatments for the associated diseases.

The effects of a normal and a low molecular weight (LMW) heparin fraction were compared by four coagulation methods. Plasma samples of patients were investigated who were treated with normal heparin or with a LMW heparin. The study was undertaken to analyze the interrelationship between the coagulation methods: Heptest, activated partial thromboplastin time, thrombin clotting time, and S 2222 chromogenic anti-factor Xa test. The results showed a high correlation between Heptest and S 2222 anti-factor Xa method for unfractionated and LMW heparin (r = 0.91 and 0.90). Comparing the coagulation times in seconds of Heptest and aPTT, the correlations were r = 0.56 (normal heparin) and 0.15 (LMW heparin). The correlation between the coagulation times of Heptest and thrombin clotting time were r = 0.65 and 0.25, respectively. The correlations between the coagulation methods were higher, when coagulation times rather than transformed values to units per liter of the Heptest and of the S 2222 anti-factor Xa method were employed. Furthermore, the data demonstrate a high sensitivity of the Heptest to conventional and LMW heparin, whereas activated partial thromboplastin time and thrombin clotting time are less sensitive to either heparin. For laboratory control of LMW heparin, Heptest and S 2222 chromogenic method are reliable tests. Therapeutic ranges will have to be established.