The small GTPase KlRho5 responds to oxidative stress and affects cytokinesis

ABSTRACT Rho5 is the yeast homolog of the human small GTPase Rac1. We characterized the genes encoding Rho5 and the subunits of its dimeric activating guanine-nucleotide-exchange factor (GEF), Dck1 and Lmo1, in the yeast Kluyveromyces lactis. Rapid translocation of the three GFP-tagged components to mitochondria upon oxidative stress and carbon starvation indicate a similar function of KlRho5 in energy metabolism and mitochondrial dynamics as described for its Saccharomyces cerevisiae homolog. Accordingly, Klrho5 deletion mutants are hyper-resistant towards hydrogen peroxide. Moreover, synthetic lethalities of rho5 deletions with key components in nutrient sensing, such as sch9 and gpr1, are not conserved in K. lactis. Instead, Klrho5 deletion mutants display morphological defects with strengthened lateral cell walls and protruding bud scars. The latter result from aberrant cytokinesis, as observed by following the budding process in vivo and by transmission electron microscopy of the bud neck region. This phenotype can be suppressed by KlCDC42G12V, which encodes a hyper-active variant. Data from live-cell fluorescence microscopy support the notion that KlRho5 interferes with the actin moiety of the contractile actomyosin ring, with consequences different from those previously reported for mutants lacking myosin.

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MITOL deletion in the brain impairs mitochondrial structure and ER tethering leading to oxidative stress

Life Science Alliance ◽

10.26508/lsa.201900308 ◽

2019 ◽

Vol 2 (4) ◽

pp. e201900308 ◽

Cited By ~ 7

Author(s):

Shun Nagashima ◽

Keisuke Takeda ◽

Nobuhiko Ohno ◽

Satoshi Ishido ◽

Motohide Aoki ◽

...

Keyword(s):

Oxidative Stress ◽

Microglial Activation ◽

Developmental Disorders ◽

Inflammatory Diseases ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Three Dimensional ◽

Abnormal Behavior ◽

Mitochondrial Abnormalities

Mitochondrial abnormalities are associated with developmental disorders, although a causal relationship remains largely unknown. Here, we report that increased oxidative stress in neurons by deletion of mitochondrial ubiquitin ligase MITOL causes a potential neuroinflammation including aberrant astrogliosis and microglial activation, indicating that mitochondrial abnormalities might confer a risk for inflammatory diseases in brain such as psychiatric disorders. A role of MITOL in both mitochondrial dynamics and ER-mitochondria tethering prompted us to characterize three-dimensional structures of mitochondria in vivo. In MITOL-deficient neurons, we observed a significant reduction in the ER-mitochondria contact sites, which might lead to perturbation of phospholipids transfer, consequently reduce cardiolipin biogenesis. We also found that branched large mitochondria disappeared by deletion of MITOL. These morphological abnormalities of mitochondria resulted in enhanced oxidative stress in brain, which led to astrogliosis and microglial activation partly causing abnormal behavior. In conclusion, the reduced ER-mitochondria tethering and excessive mitochondrial fission may trigger neuroinflammation through oxidative stress.

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Analysis of Functional Domains in Rho5, the Yeast Homolog of Human Rac1 GTPase, in Oxidative Stress Response

International Journal of Molecular Sciences ◽

10.3390/ijms20225550 ◽

2019 ◽

Vol 20 (22) ◽

pp. 5550 ◽

Cited By ~ 1

Author(s):

Carolin Sterk ◽

Lauren Gräber ◽

Hans-Peter Schmitz ◽

Jürgen J. Heinisch

Keyword(s):

Oxidative Stress ◽

Stress Response ◽

Hypervariable Region ◽

Oxidative Stress Response ◽

Small Gtpase ◽

Rac1 Gtpase ◽

Yeast Homolog ◽

And Oxidative Stress ◽

Polybasic Region

The small GTPase Rho5 of Saccharomyces cerevisiae is required for proper regulation of different signaling pathways, which includes the response to cell wall, osmotic, nutrient, and oxidative stress. We here show that proper in vivo function and intracellular distribution of Rho5 depends on its hypervariable region at the carboxyterminal end, which includes the CAAX box for lipid modification, a preceding polybasic region (PBR) carrying a serine residue, and a 98 amino acid–specific insertion only present in Rho5 of S. cerevisiae but not in its human homolog Rac1. Results from trapping GFP-Rho5 variants to the mitochondrial surface suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation to mitochondria in order to fulfil its role in oxidative stress response. These findings are supported by heterologous expression of a codon-optimized human RAC1 gene, which can only complement a yeast rho5 deletion in a chimeric fusion with RHO5 sequences that restore the correct spatiotemporal distribution of the encoded protein.

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Evidence for Two CRIB Domains in Phospholipase D2 (PLD2) That the Enzyme Uses to Specifically Bind to the Small GTPase Rac2

Journal of Biological Chemistry ◽

10.1074/jbc.m110.206672 ◽

2011 ◽

Vol 286 (18) ◽

pp. 16308-16320 ◽

Cited By ~ 19

Author(s):

Hong-Juan Peng ◽

Karen M. Henkels ◽

Madhu Mahankali ◽

Mary C. Dinauer ◽

Julian Gomez-Cambronero

Keyword(s):

Specific Binding ◽

Strong Association ◽

Living Cells ◽

Small Gtpases ◽

Small Gtpase ◽

Deletion Mutants ◽

Ph Domain ◽

Phospholipase D2

Phospholipase D (PLD) and small GTPases are vital to cell signaling. We report that the Rac2 and the PLD2 isoforms exist in the cell as a lipase-GTPase complex that enables the two proteins to elicit their respective functionalities. A strong association between the two molecules was demonstrated by co-immunoprecipitation and was confirmed in living cells by FRET with CFP-Rac2 and YFP-PLD2 fluorescent chimeras. We have identified the amino acids in PLD2 that define a specific binding site to Rac2. This site is composed of two CRIB (Cdc42-and Rac-interactive binding) motifs that we have named “CRIB-1” and “CRIB-2” in and around the PH domain in PLD2. Deletion mutants PLD2-ΔCRIB-1/2 negate co-immunoprecipitation with Rac2 and diminish the FRET signal in living cells. The PLD2-Rac2 association was further confirmed in vitro using affinity-purified recombinant proteins. Binding was saturable with an apparent Kd of 3 nm and was diminished with PLD2-ΔCRIB mutants. Furthermore, PLD2 bound more efficiently to Rac2-GTP than to Rac2-GDP or to a GDP-constitutive Rac2-N17 mutant. Increasing concentrations of recombinant Rac2 in vitro and in vivo during cell adhesion inhibit PLD2. Conversely, Rac2 activity is increased in the presence of PLD2-WT but not in PLD2-ΔCRIB. We propose that in activated cells PLD2 affects Rac2 in an initial positive feedback, but as Rac2-GTP accumulates in the cell, this constitutes a “termination signal” leading to PLD2 inactivation.

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Loss of EPAC2 results in altered synaptic composition of dendritic spines and excitatory and inhibitory balance

10.1101/526772 ◽

2019 ◽

Author(s):

Kelly A Jones ◽

Michiko Sumiya ◽

Kevin M Woolfrey ◽

Deepak P Srivastava ◽

Peter Penzes

Keyword(s):

Ampa Receptors ◽

Cortical Neurons ◽

Glutamate Transporter ◽

Autism Spectrum ◽

Small Gtpase ◽

Anterior Cingulate ◽

Synaptic Proteins ◽

Inhibitory Synapses ◽

Nucleotide Exchange

EPAC2 is a guanine nucleotide exchange factor that regulates GTPase activity of the small GTPase Rap and Ras) and is highly enriched at synapses. Activation of EPAC2 has been shown to induce dendritic spine shrinkage and increase spine motility, effects that are necessary for synaptic plasticity. These morphological effects are dysregulated by rare mutations of EPAC2 associated with autism spectrum disorders. In addition, EPAC2 destabilizes synapses through the removal of synaptic GluA2/3-containing AMPA receptors. Previous work has shown that Epac2 knockout mice (Epac2-/-) display abnormal social interactions, as well as gross disorganization of the frontal cortex and abnormal spine motility in vivo. In this study we sought to further understand the cellular consequences of knocking out Epac2 on the development of neuronal and synaptic structure and organization of cortical neurons. Using primary cortical neurons generated from Epac2+/+ or Epac2-/- mice, we confirm that EPAC2 is required for cAMP-dependent spine shrinkage. Neurons from Epac2-/- mice also displayed increased synaptic expression of GluA2/3-containing AMPA receptors, as well as of the adhesion protein N-cadherin. Intriguingly, analysis of excitatory and inhibitory synaptic proteins revealed that loss of EPAC2 resulted in altered of expression of vesicular glutamate transporter 1 (VGluT1) and vesicular GABA transporter (VGAT), indicating a potential imbalance in excitatory/inhibitory inputs onto neurons. Finally, examination of cortical neurons located within the anterior cingulate cortex further revealed subtle deficits in the establishment of dendritic arborization in vivo. These data provide evidence that EPAC2 is required for the correct composition of synapses and that loss of this protein could result in an imbalance of excitatory and inhibitory synapses.

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Abstract 392: Angiotensin II TypeI Receptor-mediated Mitochondrial Fission and Suppression of Mitophagy Play Crucial Role in Vascular Senescence and Arteriosclerosis

Circulation Research ◽

10.1161/res.127.suppl_1.392 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Yoshihiro Uchikado ◽

Yoshiyuki Ikeda ◽

Yuichi Sasaki ◽

Yuichi Akasaki ◽

Mitsuru Ohishi

Keyword(s):

Oxidative Stress ◽

Angiotensin Ii ◽

Mitochondrial Dysfunction ◽

Cellular Senescence ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Double Knockout ◽

Apoe Ko ◽

Vascular Senescence

Introduction: Metabolic stress including oxidized low density lipoprotein (ox-LDL) cause mitochondrial dysfunction and evoke vascular senescence and atherosclerosis. Mitochondria are highly dynamic organelles that undergo quality control by mitochondrial dynamics and mitophagy. This study aims to clarify whether and how mitochondrial dynamics and mitophagy are involved in the etiology of vascular senescence and arteriosclerosis. Methods: VSMC were stimulated by ox-LDL. We also conducted in vivo experiment using C57BL6 (WT), apolipoprotein E (ApoE) deficient and the double knockout of ApoE mice and Angiotensin II Type1 Receptor (AT1R). Results: Treatment of ox-LDL forced mitochondria to fission through activation of Drp1, induced mitochondrial dysfunction and oxidative stress, and developed cellular senescence. Inhibition of either Drp1, AT1R, MAPK retarded them, suggesting that mitochondrial fission plays key roles to develop premature cellular senescence and is modulated by AT1R/MAPK signal.Administration of ox-LDL decreased the number of mitophagy assessed by electron microscopy and immunohistochemistry of LAMP2 and TOMM20. AT1R signal inhibition increased mitophagy which was not affected by Atg7 knockdown, whereas it was decreased by either Rab9 or Ulk1 knockdown. Immunohistochemistry showed Rab9 dots were co-localized to TOMM20 and LAMP2, whereas LC3 dots were not, suggesting that AT1R signal induces mitophagy through Rab9-dependent alternative autophagy. The degree of vascular senescence was higher, the number of fused mitochondria and mitochondrial function were lower and mitochondrial oxidative stress was higher in ApoE KO than those in WT. DKO attenuated these adverse effect of ApoE KO. Conclusion: AT1R regulates vascular senescence and arteriosclerosis via induction of mitochondrial fission and inhibition of mitophagy.

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Attachment of the Ubiquitin-Related Protein Urm1p to the Antioxidant Protein Ahp1p

Eukaryotic Cell ◽

10.1128/ec.2.5.930-936.2003 ◽

2003 ◽

Vol 2 (5) ◽

pp. 930-936 ◽

Cited By ~ 71

Author(s):

April S. Goehring ◽

David M. Rivers ◽

George F. Sprague

Keyword(s):

Oxidative Stress ◽

Saccharomyces Cerevisiae ◽

Stress Response ◽

Posttranslational Modification ◽

Oxidative Stress Response ◽

Nutrient Sensing ◽

Related Protein ◽

Antioxidant Protein ◽

Antioxidant Stress

ABSTRACT Urm1p is a ubiquitin-related protein that serves as a posttranslational modification of other proteins. Urm1p conjugation has been implicated in the budding process and in nutrient sensing. Here, we have identified the first in vivo target for the urmylation pathway as the antioxidant protein Ahp1p. The attachment of Urm1p to Ahp1p requires the E1 for the urmylation pathway, Uba4p. Loss of the urmylation pathway components results in sensitivity to a thiol-specific oxidant, as does loss of Ahp1p, implying that urmylation has a role in an oxidative-stress response. Moreover, treatment of cells with thiol-specific oxidants affects the abundance of Ahp1p-Urm1p conjugates. These results suggest that the conjugation of Urm1p to Ahp1p could regulate the function of Ahp1p in antioxidant stress response in Saccharomyces cerevisiae.

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In Vivo Dynamics of Rac-Membrane Interactions

Molecular Biology of the Cell ◽

10.1091/mbc.e06-01-0005 ◽

2006 ◽

Vol 17 (6) ◽

pp. 2770-2779 ◽

Cited By ~ 67

Author(s):

Konstadinos Moissoglu ◽

Boris M. Slepchenko ◽

Nahum Meller ◽

Alan F. Horwitz ◽

Martin A. Schwartz

Keyword(s):

Dissociation Rate ◽

Small Gtpase ◽

Nucleotide Exchange ◽

Hairpin Rna ◽

Nucleotide Exchange Factors ◽

Gtpase Activating Proteins ◽

Detectable Rate ◽

Major Route ◽

Membrane Bound

The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (koff) of membrane-bound GFP-Rac. We find that koff is 0.048 s−1 for wtRac and ∼10-fold less (0.004 s−1) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased koff for wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on koff. These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.

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CalDAG-GEFI deficiency protects mice in a novel model of FcγRIIA-mediated thrombosis and thrombocytopenia

Blood ◽

10.1182/blood-2011-03-342352 ◽

2011 ◽

Vol 118 (4) ◽

pp. 1113-1120 ◽

Cited By ~ 45

Author(s):

Moritz Stolla ◽

Lucia Stefanini ◽

Pierrette André ◽

Timothy D. Ouellette ◽

Michael P. Reilly ◽

...

Keyword(s):

Platelet Activation ◽

Near Infrared ◽

Infrared Imaging ◽

Small Gtpase ◽

In Vivo Studies ◽

Critical Event ◽

Nucleotide Exchange ◽

P2y12 Inhibitor ◽

Novel Model

AbstractPlatelet activation via Fcγ receptor IIA (FcγRIIA) is a critical event in immune-mediated thrombocytopenia and thrombosis syndromes (ITT). We recently identified signaling by the guanine nucleotide exchange factor CalDAG-GEFI and the adenosine diphosphate receptor P2Y12 as independent pathways leading to Rap1 small GTPase activation and platelet aggregation. Here, we evaluated the contribution of CalDAG-GEFI and P2Y12 signaling to platelet activation in ITT. Mice transgenic for the human FcγRIIA (hFcR) and deficient in CalDAG-GEFI−/− (hFcR/CDGI−/−) were generated. Compared with controls, aggregation of hFcR/CDGI−/− platelets or P2Y12 inhibitor-treated hFcR platelets required more than 5-fold and approximately 2-fold higher concentrations of a FcγRIIA stimulating antibody against CD9, respectively. Aggregation and Rap1 activation were abolished in P2Y12 inhibitor-treated hFcR/CDGI−/− platelets. For in vivo studies, a novel model for antibody-induced thrombocytopenia and thrombosis was established. FcγRIIA-dependent platelet thrombosis was induced by infusion of Alexa750-labeled antibodies to glycoprotein IX (CD42a), and pulmonary thrombi were detected by near-infrared imaging technology. Anti-GPIX antibodies dose-dependently caused thrombocytopenia and pulmonary thrombosis in hFcR-transgenic but not wild-type mice. CalDAG-GEFI-deficient but not clopidogrel-treated hFcR-transgenic mice were completely protected from ITT. In summary, we established a novel mouse model for ITT, which was used to identify CalDAG-GEFI as a potential new target in the treatment of ITT.

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COPI Recruitment Is Modulated by a Rab1b-dependent Mechanism

Molecular Biology of the Cell ◽

10.1091/mbc.e02-09-0625 ◽

2003 ◽

Vol 14 (5) ◽

pp. 2116-2127 ◽

Cited By ~ 86

Author(s):

Cecilia Alvarez ◽

Rafael Garcia-Mata ◽

Elizabeth Brandon ◽

Elizabeth Sztul

Keyword(s):

Active Form ◽

Brefeldin A ◽

Small Gtpase ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Inactive Form ◽

Exact Function ◽

Guanine Nucleotide Binding

The small GTPase Rab1b is essential for endoplasmic reticulum (ER) to Golgi transport, but its exact function remains unclear. We have examined the effects of wild-type and three mutant forms of Rab1b in vivo. We show that the inactive form of Rab1b (the N121I mutant with impaired guanine nucleotide binding) blocks forward transport of cargo and induces Golgi disruption. The phenotype is analogous to that induced by brefeldin A (BFA): it causes resident Golgi proteins to relocate to the ER and induces redistribution of ER-Golgi intermediate compartment proteins to punctate structures. The COPII exit machinery seems to be functional in cells expressing the N121I mutant, but COPI is compromised, as shown by the release of β-COP into the cytosol. Our results suggest that Rab1b function influences COPI recruitment. In support of this, we show that the disruptive effects of N121I can be reversed by expressing known mediators of COPI recruitment, the GTPase ARF1 and its guanine nucleotide exchange factor GBF1. Further evidence is provided by the finding that cells expressing the active form of Rab1b (the Q67L mutant with impaired GTPase activity) are resistant to BFA. Our data suggest a novel role for Rab1b in ARF1- and GBF1-mediated COPI recruitment pathway.

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Ras recruits mitotic exit regulator Lte1 to the bud cortex in budding yeast

The Journal of Cell Biology ◽

10.1083/jcb.200301128 ◽

2003 ◽

Vol 161 (5) ◽

pp. 889-897 ◽

Cited By ~ 46

Author(s):

Satoshi Yoshida ◽

Ryuji Ichihashi ◽

Akio Toh-e

Keyword(s):

Small Gtpase ◽

Mitotic Exit ◽

Effector Protein ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Downstream Effector ◽

Nucleotide Exchange Factor ◽

Family Protein

ACdc25 family protein Lte1 (low temperature essential) is essential for mitotic exit at a lowered temperature and has been presumed to be a guanine nucleotide exchange factor (GEF) for a small GTPase Tem1, which is a key regulator of mitotic exit. We found that Lte1 physically associates with Ras2-GTP both in vivo and in vitro and that the Cdc25 homology domain (CHD) of Lte1 is essential for the interaction with Ras2. Furthermore, we found that the proper localization of Lte1 to the bud cortex is dependent on active Ras and that the overexpression of a derivative of Lte1 without the CHD suppresses defects in mitotic exit of a Δlte1 mutant and a Δras1 Δras2 mutant. These results suggest that Lte1 is a downstream effector protein of Ras in mitotic exit and that the Ras GEF domain of Lte1 is not essential for mitotic exit but required for its localization.

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