Fission yeast NDR/LATS kinase Orb6 regulates exocytosis via phosphorylation of the exocyst complex (original) (raw)
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Journal of Cell Biology, 2001
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functi...
The Journal of Cell Biology, 2005
ho GTPases are important regulators of polarity in eukaryotic cells. In yeast they are involved in regulating the docking and fusion of secretory vesicles with the cell surface. Our analysis of a Rho3 mutant that is unable to interact with the Exo70 subunit of the exocyst reveals a normal polarization of the exocyst complex as well as other polarity markers. We also find that there is no redundancy between the Rho3-Exo70 and Rho1-Sec3 pathways in the localization of the exocyst. This R suggests that Rho3 and Cdc42 act to polarize exocytosis by activating the exocytic machinery at the membrane without the need to first recruit it to sites of polarized growth. Consistent with this model, we find that the ability of Rho3 and Cdc42 to hydrolyze GTP is not required for their role in secretion. Moreover, our analysis of the Sec3 subunit of the exocyst suggests that polarization of the exocyst may be a consequence rather than a cause of polarized exocytosis.
Spatial regulation of exocytosis and cell polarity: Yeast as a model for animal cells
FEBS Letters, 2007
Exocytosis is the major mechanism by which new membrane components are delivered to the cell surface. In most, if not all, eukaryotic cells this is also a highly spatially regulated process that is tightly coordinated with the overall polarity of a cell. The Rho/Cdc42 family of GTPases and the lethal giant larvae/Sro7 family are two highly conserved families of proteins which appear to have dual functions both in cell polarity and exocytosis. Analysis of their functions has begun to unravel the coordination between these processes and propose a model for polarized vesicle docking and fusion at the site of asymmetric cell growth.
Journal of Cell Science, 2011
Although endocytosis and exocytosis have been extensively studied in budding yeast, there have been relatively few investigations of these complex processes in the fission yeast Schizosaccharomyces pombe. Here we identify and characterize fission yeast Mug33, a novel Tea1-interacting protein, and show that Mug33 is involved in exocytosis. Mug33 is a Sur7/PalI-family transmembrane protein that localizes to the plasma membrane at the cell tips and to cytoplasmic tubulovesicular elements (TVEs). A subset of Mug33 TVEs make long-range movements along actin cables, co-translocating with subunits of the exocyst complex. TVE movement depends on the type V myosin Myo52. Although mug33 mutants are viable, with only a mild cell-polarity phenotype, mug33 myo52 double mutants are synthetically lethal. Combining mug33 with deletion of the formin For3 (for3) leads to synthetic temperature-sensitive growth and strongly reduced levels of exocytosis. Interestingly, mutants in non-essential genes involved in exocyst function behave in a manner similar to mug33 when combined with myo52 and for3. By contrast, combining mug33 with mutants in non-essential exocyst genes has only minor effects on growth. We propose that Mug33 contributes to exocyst function and that actin cable-dependent vesicle transport and exocyst function have complementary roles in promoting efficient exocytosis in fission yeast.
Membrane targeting of the yeast exocyst complex
Biochimica et biophysica acta, 2015
The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits - Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP2) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP2. We found that PIP2 is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into un...
Journal of Biological Chemistry, 2003
In the fission yeast Schizosaccharomyces pombe, proper establishment and maintenance of cell polarity require Orb6p, a highly conserved serine/threonine kinase involved in regulating both cell morphogenesis and cell cycle control. Orb6p localizes to the cell tips during interphase and to the cell septum during mitosis. To investigate the mechanisms involved in Orb6p function, we conducted a two-hybrid screen to identify proteins that interact with Orb6p. Using this approach, we identified Skb1p, a highly conserved protein methyltransferase that has been implicated previously in cell cycle control, in the coordination of cell cycle progression with morphological changes, and in hyperosmotic stress response. We found that Skb1p associates with Orb6p in S. pombe cells and that the two proteins interact directly in vitro. Loss of Skb1p exacerbates the phenotype of orb6 mutants, suggesting that Skb1p and Orb6p functionally interact in S. pombe cells. Our results suggest that Skb1p affects the intracellular localization of Orb6p and that loss of Skb1p leads to a redistribution of the Orb6p kinase away from the cell tips. Furthermore, we found that Orb6p kinase activity is strongly increased following exposure to salt shock, suggesting that Orb6p has a role in cell response to hyperosmotic stress. Previous studies have shown that Skb1p interacts with the fission yeast p21-activated kinase homologue Pak1p/Shk1p to regulate cell polarity and cell cycle progression. Our findings identify Orb6p as an additional target for Skb1p and suggest a novel function for Skb1p in the control of cell polarity by regulating the subcellular localization of Orb6p.
Proceedings of the National …, 1998
The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine͞threonine protein kinase, belonging to the myotonic dystrophy kinase͞ cot1͞warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34 cdc2 mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1͞Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1͞Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Molecular Biology of the Cell, 2009
The Rho3 and Cdc42 members of the Rho GTPase family are important regulators of exocytosis in yeast. However, the precise mechanism by which they regulate this process is controversial. Here, we present evidence that the Exo70 component of the exocyst complex is a direct effector of both Rho3 and Cdc42. We identify gain-of-function mutants in EXO70 that potently suppress mutants in RHO3 and CDC42 defective for exocytic function. We show that Exo70 has the biochemical properties expected of a direct effector for both Rho3 and Cdc42. Surprisingly, we find that C-terminal prenylation of these GTPases both promotes the interaction and influences the sites of binding within Exo70. Finally, we demonstrate that the phenotypes associated with novel loss-of-function mutants in EXO70, are entirely consistent with Exo70 as an effector for both Rho3 and Cdc42 function in secretion. These data suggest that interaction with the Exo70 component of the exocyst is a key event in spatial regulation o...
Cdc42 Interacts with the Exocyst and Regulates Polarized Secretion
Journal of Biological Chemistry, 2001
Polarized delivery and incorporation of proteins and lipids to specific domains of the plasma membrane is fundamental to a wide range of biological processes such as neuronal synaptogenesis and epithelial cell polarization. The exocyst complex is specifically localized to sites of active exocytosis and plays essential roles in secretory vesicle targeting and docking at the plasma membrane. Sec3p, a component of the exocyst, is thought to be a spatial landmark for polarized exocytosis. In a search for proteins that regulate the localization of the exocyst in the budding yeast Saccharomyces cerevisiae, we found that certain cdc42 mutants affect the polarized localization of the exocyst proteins. In addition, we found that these mutant cells have a randomized protein secretion pattern on the cell surface. Biochemical experiments indicated that Sec3p directly interacts with Cdc42 in its GTP-bound form. Genetic studies demonstrated synthetically lethal interactions between cdc42 and several exocyst mutants. These results have revealed a role for Cdc42 in exocytosis. We propose that Cdc42 coordinates the vesicle docking machinery and the actin cytoskeleton for polarized secretion.