scholarly journals Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex

2005 ◽  
Vol 170 (4) ◽  
pp. 583-594 ◽  
Author(s):  
Olivier Roumanie ◽  
Hao Wu ◽  
Jeffrey N. Molk ◽  
Guendalina Rossi ◽  
Kerry Bloom ◽  
...  
Keyword(s):  
Cell Surface ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Eukaryotic Cells ◽  
Secretory Vesicles ◽  
Polarized Growth ◽  
Gtp Hydrolysis ◽  
Exocyst Complex ◽  
Polarized Exocytosis

Rho 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 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.

scholarly journals The Exo70 Subunit of the Exocyst Is an Effector for Both Cdc42 and Rho3 Function in Polarized Exocytosis

2010 ◽  
Vol 21 (3) ◽  
pp. 430-442 ◽  
Author(s):  
Hao Wu ◽  
Courtney Turner ◽  
Jimmy Gardner ◽  
Brenda Temple ◽  
Patrick Brennwald
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Biochemical Properties ◽  
Loss Of Function ◽  
Gain Of Function ◽  
Present Evidence ◽  
Spatial Regulation ◽  
Exocyst Complex ◽  
Polarized Exocytosis

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 of exocytosis by Rho GTPases.

scholarly journals The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth

2014 ◽  
Vol 25 (8) ◽  
pp. 1312-1326 ◽  
Author(s):  
Meritxell Riquelme ◽  
Erin L. Bredeweg ◽  
Olga Callejas-Negrete ◽  
Robert W. Roberson ◽  
Sarah Ludwig ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Hyphal Growth ◽  
Apical Plasma Membrane ◽  
Apical Region ◽  
Secretory Vesicles ◽  
Polarized Growth ◽  
Hyphal Morphogenesis ◽  
Exocyst Complex ◽  
Dynamic Formation

Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.

scholarly journals Identifying Cancer-Relevant Mutations in the DLC START Domain Using Evolutionary and Structure-Function Analyses

2020 ◽  
Vol 21 (21) ◽  
pp. 8175
Author(s):  
Ashton S. Holub ◽  
Renee A. Bouley ◽  
Ruben C. Petreaca ◽  
Aman Y. Husbands
Keyword(s):  
Structure Function ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Structural Level ◽  
Lipid Transfer ◽  
Gtp Hydrolysis ◽  
Conserved Residues ◽  
Wide Range ◽  
Bona Fide ◽  
Start Domain

Rho GTPase signaling promotes proliferation, invasion, and metastasis in a broad spectrum of cancers. Rho GTPase activity is regulated by the deleted in liver cancer (DLC) family of bona fide tumor suppressors which directly inactivate Rho GTPases by stimulating GTP hydrolysis. In addition to a RhoGAP domain, DLC proteins contain a StAR-related lipid transfer (START) domain. START domains in other organisms bind hydrophobic small molecules and can regulate interacting partners or co-occurring domains through a variety of mechanisms. In the case of DLC proteins, their START domain appears to contribute to tumor suppressive activity. However, the nature of this START-directed mechanism, as well as the identities of relevant functional residues, remain virtually unknown. Using the Catalogue of Somatic Mutations in Cancer (COSMIC) dataset and evolutionary and structure-function analyses, we identify several conserved residues likely to be required for START-directed regulation of DLC-1 and DLC-2 tumor-suppressive capabilities. This pan-cancer analysis shows that conserved residues of both START domains are highly overrepresented in cancer cells from a wide range tissues. Interestingly, in DLC-1 and DLC-2, three of these residues form multiple interactions at the tertiary structural level. Furthermore, mutation of any of these residues is predicted to disrupt interactions and thus destabilize the START domain. As such, these mutations would not have emerged from traditional hotspot scans of COSMIC. We propose that evolutionary and structure-function analyses are an underutilized strategy which could be used to unmask cancer-relevant mutations within COSMIC. Our data also suggest DLC-1 and DLC-2 as high-priority candidates for development of novel therapeutics that target their START domain.

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells

2005 ◽  
Vol 288 (6) ◽  
pp. C1411-C1419 ◽  
Author(s):  
Bo Wang ◽  
Fiona G. Wylie ◽  
Rohan D. Teasdale ◽  
Jennifer L. Stow
Keyword(s):  
Cell Surface ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Dominant Negative ◽  
Adhesion Protein ◽  
Cell Functions ◽  
Golgi Transport ◽  
Polarized Trafficking ◽  
Basolateral Trafficking ◽  
E Cadherin

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120 ctn interacts with E-cadherin, because p120 ctn localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin.

scholarly journals Control of Polarized Growth by the Rho Family GTPase Rho4 in Budding Yeast: Requirement of the N-Terminal Extension of Rho4 and Regulation by the Rho GTPase-Activating Protein Bem2

2012 ◽  
Vol 12 (2) ◽  
pp. 368-377 ◽  
Author(s):  
Ting Gong ◽  
Yuan Liao ◽  
Fei He ◽  
Yang Yang ◽  
Dan-Dan Yang ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Budding Yeast ◽  
Bimolecular Fluorescence Complementation ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Polarized Growth ◽  
Gtpase Activating Protein ◽  
Content Type ◽  
Morphological Defects

ABSTRACT In the budding yeast Saccharomyces cerevisiae , Rho4 GTPase partially plays a redundant role with Rho3 in the control of polarized growth, as deletion of RHO4 and RHO3 together, but not RHO4 alone, caused lethality and a loss of cell polarity at 30°C. Here, we show that overexpression of the constitutively active rho4 Q131L mutant in an rdi1 Δ strain caused a severe growth defect and generated large, round, unbudded cells, suggesting that an excess of Rho4 activity could block bud emergence. We also generated four temperature-sensitive rho4-Ts alleles in a rho3 Δ rho4 Δ strain. These mutants showed growth and morphological defects at 37°C. Interestingly, two rho4-Ts alleles contain mutations that cause amino acid substitutions in the N-terminal region of Rho4. Rho4 possesses a long N-terminal extension that is unique among the six Rho GTPases in the budding yeast but is common in Rho4 homologs in other yeasts and filamentous fungi. We show that the N-terminal extension plays an important role in Rho4 function since rho3 Δ rho4 Δ 61 cells expressing truncated Rho4 lacking amino acids (aa) 1 to 61 exhibited morphological defects at 24°C and a growth defect at 37°C. Furthermore, we show that Rho4 interacts with Bem2, a Rho GTPase-activating protein (RhoGAP) for Cdc42 and Rho1, by yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and glutathione S -transferase (GST) pulldown assays. Bem2 specifically interacts with the GTP-bound form of Rho4, and the interaction is mediated by its RhoGAP domain. Overexpression of BEM2 aggravates the defects of rho3 Δ rho4 mutants. These results suggest that Bem2 might be a novel GAP for Rho4.

scholarly journals Rho GTPases: regulation of cell polarity and growth in yeasts

2010 ◽  
Vol 426 (3) ◽  
pp. 243-253 ◽  
Author(s):  
Pilar Perez ◽  
Sergio A. Rincón
Keyword(s):  
Cell Wall ◽  
Cell Polarity ◽  
Rho Gtpases ◽  
Molecular Mechanisms ◽  
Eukaryotic Cells ◽  
Signalling Pathways ◽  
Polarized Growth ◽  
Cell Integrity ◽  
Actin Organization ◽  
Wide Range

Eukaryotic cells display a wide range of morphologies important for cellular function and development. A particular cell shape is made via the generation of asymmetry in the organization of cytoskeletal elements, usually leading to actin localization at sites of growth. The Rho family of GTPases is present in all eukaryotic cells, from yeast to mammals, and their role as key regulators in the signalling pathways that control actin organization and morphogenetic processes is well known. In the present review we will discuss the role of Rho GTPases as regulators of yeasts' polarized growth, their mechanism of activation and signalling pathways in Saccharomyces cerevisiae and Schizosaccharomyces pombe. These two model yeasts have been very useful in the study of the molecular mechanisms responsible for cell polarity. As in other organisms with cell walls, yeast's polarized growth is closely related to cell-wall biosynthesis, and Rho GTPases are critical modulators of this process. They provide the co-ordinated regulation of cell-wall biosynthetic enzymes and actin organization required to maintain cell integrity during vegetative growth.

Immunogold and immunoblot studies on a cell surface protein found in primary mesenchyme cells and in the cortical secretory vesicles of the sea urchin egg

1987 ◽  
Vol 45 ◽  
pp. 832-833
Author(s):  
G.L. Decker ◽  
M.C. Valdizan
Keyword(s):  
Cell Surface ◽  
Sea Urchin ◽  
Surface Protein ◽  
Egg Cell ◽  
Secretory Vesicles ◽  
Cell Surface Protein ◽  
Surface Complexes ◽  
Mesenchyme Cells ◽  
Lowicryl K4m ◽  
Primary Mesenchyme Cells

A monoclonal antibody designated MAb 1223 has been used to show that primary mesenchyme cells of the sea urchin embryo express a 130-kDa cell surface protein that may be directly involved in Ca2+ uptake required for growth of skeletal spicules. Other studies from this laboratory have shown that the 1223 antigen, although in relatively low abundance, is also expressed on the cell surfaces of unfertilized eggs and on the majority of blastomeres formed prior to differentiation of the primary mesenchyme cells.We have studied the distribution of 1223 antigen in S. purpuratus eggs and embryos and in isolated egg cell surface complexes that contain the cortical secretory vesicles. Specimens were fixed in 1.0% paraformaldehyde and 1.0% glutaraldehyde and embedded in Lowicryl K4M as previously reported. Colloidal gold (8nm diameter) was prepared by the method of Mulpfordt.

scholarly journals Regulation of Rho GTPases by RhoGDIs in Human Cancers

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1037 ◽  
Author(s):  
Cho ◽  
Kim ◽  
Baek ◽  
Kim ◽  
Lee
Keyword(s):  
Cell Migration ◽  
Cancer Progression ◽  
Anticancer Agents ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Regulatory Mechanisms ◽  
Malignant Phenotype ◽  
Cellular Processes ◽  
Human Cancers

Rho GDP dissociation inhibitors (RhoGDIs) play important roles in various cellular processes, including cell migration, adhesion, and proliferation, by regulating the functions of the Rho GTPase family. Dissociation of Rho GTPases from RhoGDIs is necessary for their spatiotemporal activation and is dynamically regulated by several mechanisms, such as phosphorylation, sumoylation, and protein interaction. The expression of RhoGDIs has changed in many human cancers and become associated with the malignant phenotype, including migration, invasion, metastasis, and resistance to anticancer agents. Here, we review how RhoGDIs control the function of Rho GTPases by regulating their spatiotemporal activity and describe the regulatory mechanisms of the dissociation of Rho GTPases from RhoGDIs. We also discuss the role of RhoGDIs in cancer progression and their potential uses for therapeutic intervention.

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