Article

The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity.

Department of Cell Biology, Cell Biology, and Genetics, Weill Medical College of Cornell University, New York, New York 10021, USA.
Molecular Biology of the Cell (Impact Factor: 4.55). 01/2000; 10(12):4121-33. DOI: 10.1091/mbc.10.12.4121
Source: PubMed

ABSTRACT Budding yeast grow asymmetrically by the polarized delivery of proteins and lipids to specific sites on the plasma membrane. This requires the coordinated polarization of the actin cytoskeleton and the secretory apparatus. We identified Rho3 on the basis of its genetic interactions with several late-acting secretory genes. Mutational analysis of the Rho3 effector domain reveals three distinct functions in cell polarity: regulation of actin polarity, transport of exocytic vesicles from the mother cell to the bud, and docking and fusion of vesicles with the plasma membrane. We provide evidence that the vesicle delivery function of Rho3 is mediated by the unconventional myosin Myo2 and that the docking and fusion function is mediated by the exocyst component Exo70. These data suggest that Rho3 acts as a key regulator of cell polarity and exocytosis, coordinating several distinct events for delivery of proteins to specific sites on the cell surface.

0 Followers
 · 
71 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers which tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here we describe a novel assay which recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 which results in a protein which is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9 which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 11/2014; 290(1). DOI:10.1074/jbc.M114.595892 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1-Rho5) and seven Rho GEFs (Scd1, Rgf1-Rgf3, and Gef1-Gef3). The GEFs for Rho2-Rho5 have not been unequivocally assigned. Particularly, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors, glucanases Eng1 and Agn1, are abnormal; and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together, our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast. © 2014 by The American Society for Cell Biology.
    Molecular Biology of the Cell 11/2014; 26(2). DOI:10.1091/mbc.E14-07-1196 · 4.55 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Small GTPases of the Rho family play an important role in regulating biological processes in fungi. In this study, we mainly investigated the biological functions of Rho3 in Botrytis cinerea, and found that deletion of the rho3 from B. cinerea significantly suppressed vegetative growth and conidiation, reduced appressorium formation and decreased virulence. Microscopy analysis revealed that the distance between septa was increased in the Δrho3 mutant. In addition, mitochondria were suggested to be the main sources of intracellular reactive oxygen species (ROS) in B. cinerea based on dual staining with 2’, 7’-dichlorodihydrofluorescein diacetate and MitoTracker orange. The Δrho3 mutant showed less accumulation of ROS in the hyphae tips compared to the WT strain of B. cinerea. These results provide the novel evidence to ascertain the function of small GTPase Rho3 in regulating growth, conidiation and virulence of B. cinerea.
    Fungal Genetics and Biology 01/2015; 75. DOI:10.1016/j.fgb.2015.01.007 · 3.26 Impact Factor

Full-text (2 Sources)

Download
4 Downloads
Available from
Dec 21, 2014