The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae

To begin to understand mechanistic differences in endocytosis in neurons and nonneuronal cells, we have compared the biochemical properties of the ubiquitously expressed dynamin-II isoform with those of neuron-specific dynamin-I. Like dynamin-I, dynamin-II is specifically localized to and highly concentrated in coated pits on the plasma membrane and can assemble in vitro into rings and helical arrays. As expected, the two closely related isoforms share a similar mechanism for GTP hydrolysis: both are stimulated in vitro by self-assembly and by interaction with microtubules or the SH3 domain-containing protein, grb2. Deletion of the C-terminal proline/arginine-rich domain from either isoform abrogates self-assembly and assembly-dependent increases in GTP hydrolysis. However, dynamin-II exhibits a ∼threefold higher rate of intrinsic GTP hydrolysis and higher affinity for GTP than dynamin-I. Strikingly, the stimulated GTPase activity of dynamin-II can be >40-fold higher than dynamin-I, due principally to its greater propensity for self-assembly and the increased resistance of assembled dynamin-II to GTP-triggered disassembly. These results are consistent with the hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.

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Intrinsic GTPase activity of a ribosomal maturation protein CgtA is associated with its inter-domain movement: insights from MD simulations and biochemical studies

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2016.1224732 ◽

2016 ◽

Vol 35 (12) ◽

pp. 2578-2587 ◽

Cited By ~ 3

Author(s):

Ananya Chatterjee ◽

Partha P. Datta

Keyword(s):

Md Simulations ◽

Gtpase Activity ◽

Domain Movement ◽

Biochemical Studies ◽

Maturation Protein ◽

Intrinsic Gtpase Activity

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The β-Subunit of the Signal Recognition Particle Receptor Is a Novel GTP-binding Protein without Intrinsic GTPase Activity

Journal of Biological Chemistry ◽

10.1074/jbc.m302158200 ◽

2003 ◽

Vol 278 (30) ◽

pp. 27712-27720 ◽

Cited By ~ 13

Author(s):

Kyle R. Legate ◽

David W. Andrews

Keyword(s):

Binding Protein ◽

Signal Recognition Particle ◽

Signal Recognition ◽

Gtpase Activity ◽

Β Subunit ◽

Gtp Binding Protein ◽

Gtp Binding ◽

Signal Recognition Particle Receptor ◽

Intrinsic Gtpase Activity

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Regulation of Cdc42 GTPase Activity in the Formation of Hyphae inCandida albicans

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0411 ◽

2007 ◽

Vol 18 (1) ◽

pp. 265-281 ◽

Cited By ~ 60

Author(s):

Helen Court ◽

Peter Sudbery

Keyword(s):

Fungal Pathogen ◽

Germ Tube ◽

Gtpase Activity ◽

Negative Regulators ◽

Human Fungal Pathogen ◽

Gtpase Activating Proteins ◽

Characteristic Features ◽

Increased Activity ◽

Intrinsic Gtpase Activity ◽

Incandida Albicans

The human fungal pathogen Candida albicans can switch between yeast, pseudohyphal, and hyphal morphologies. To investigate whether the distinctive characteristics of hyphae are due to increased activity of the Cdc42 GTPase, strains lacking negative regulators of Cdc42 were constructed. Unexpectedly, the deletion of the Cdc42 Rho guanine dissociation inhibitor RDI1 resulted in reduced rather than enhanced polarized growth. However, when cells lacking both Cdc42 GTPase-activating proteins, encoded by RGA2 and BEM3, were grown under pseudohyphal-promoting conditions the bud was highly elongated and lacked a constriction at its base, so that its shape resembled a hyphal germ tube. Moreover, a Spitzenkörper was present at the bud tip, a band of disorganized septin was present at bud base, true septin rings formed within the bud, and nuclei migrated out of the mother cell before the first mitosis. These are all characteristic features of a hyphal germ tube. Intriguingly, we observed hyphal-specific phosphorylation of Rga2, suggesting a possible mechanism for Cdc42 activation during normal hyphal development. In contrast, expression of Cdc42G12V, which is constitutively GTP bound because it lacks GTPase activity, resulted in swollen cells with prominent and stable septin bars. These results suggest the development of hyphal-specific characteristics is promoted by Cdc42-GTP in a process that also requires the intrinsic GTPase activity of Cdc42.

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GTP Hydrolysis by eRF3 Facilitates Stop Codon Decoding during Eukaryotic Translation Termination

Molecular and Cellular Biology ◽

10.1128/mcb.24.17.7769-7778.2004 ◽

2004 ◽

Vol 24 (17) ◽

pp. 7769-7778 ◽

Cited By ~ 120

Author(s):

Joe Salas-Marco ◽

David M. Bedwell

Keyword(s):

Saccharomyces Cerevisiae ◽

Polypeptide Chain ◽

Stop Codon ◽

Translation Termination ◽

Gtpase Activity ◽

Functional Importance ◽

Gtp Hydrolysis ◽

Nascent Polypeptide ◽

Eukaryotic Translation ◽

Termination Process

ABSTRACT Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3. eRF1 recognizes each of the three stop codons (UAG, UAA, and UGA) and facilitates release of the nascent polypeptide chain. eRF3 is a GTPase that stimulates the translation termination process by a poorly characterized mechanism. In this study, we examined the functional importance of GTP hydrolysis by eRF3 in Saccharomyces cerevisiae. We found that mutations that reduced the rate of GTP hydrolysis also reduced the efficiency of translation termination at some termination signals but not others. As much as a 17-fold decrease in the termination efficiency was observed at some tetranucleotide termination signals (characterized by the stop codon and the first following nucleotide), while no effect was observed at other termination signals. To determine whether this stop signal-dependent decrease in the efficiency of translation termination was due to a defect in either eRF1 or eRF3 recycling, we reduced the level of eRF1 or eRF3 in cells by expressing them individually from the CUP1 promoter. We found that the limitation of either factor resulted in a general decrease in the efficiency of translation termination rather than a decrease at a subset of termination signals as observed with the eRF3 GTPase mutants. We also found that overproduction of eRF1 was unable to increase the efficiency of translation termination at any termination signals. Together, these results suggest that the GTPase activity of eRF3 is required to couple the recognition of translation termination signals by eRF1 to efficient polypeptide chain release.

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Regulation of mitochondrial fission by GIPC-mediated Drp1 retrograde transport

Molecular Biology of the Cell ◽

10.1091/mbc.e21-06-0286 ◽

2021 ◽

Author(s):

Aaron Ramonett ◽

Eun-A Kwak ◽

Tasmia Ahmed ◽

Paola Cruz Flores ◽

Hannah R. Ortiz ◽

...

Keyword(s):

Cancer Progression ◽

Metabolic Disorders ◽

Mitochondrial Fission ◽

Retrograde Transport ◽

Binding Motif ◽

Cell Periphery ◽

Gtpase Activity ◽

Migratory Capacity ◽

Essential Enzyme ◽

Intrinsic Gtpase Activity

Drp1 is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GIPC mediates the actin-based retrograde transport of Drp1 towards the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.

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Drosophila Orc6 Facilitates GTPase Activity and Filament Formation of the Septin Complex

Molecular Biology of the Cell ◽

10.1091/mbc.e08-07-0754 ◽

2009 ◽

Vol 20 (1) ◽

pp. 270-281 ◽

Cited By ~ 30

Author(s):

Richard P.H. Huijbregts ◽

Anton Svitin ◽

Monica W. Stinnett ◽

Matthew B. Renfrow ◽

Igor Chesnokov

Keyword(s):

Origin Recognition Complex ◽

Complex Function ◽

Coiled Coil ◽

Active Role ◽

Gtpase Activity ◽

Filament Formation ◽

Cell Functions ◽

Coiled Coil Domain ◽

Intrinsic Gtpase Activity ◽

Drosophila Embryos

The origin recognition complex or ORC is a six-subunit protein important for DNA replication and other cell functions. Orc6, the smallest subunit of ORC, is essential for both replication and cytokinesis in Drosophila, and interacts with the septin protein Pnut, which is part of the Drosophila septin complex. In this study, we describe the analysis of the interaction of Orc6 with Pnut and whole Drosophila septin complex. Septin complex was purified from Drosophila embryos and also reconstituted from recombinant proteins. The interaction of Orc6 with the septin complex is dependent on the coiled-coil domain of Pnut. Furthermore, the binding of Orc6 to Pnut increases the intrinsic GTPase activity of the Drosophila septin complex, whereas in the absence of GTP it enhances septin complex filament formation. These results suggest an active role for Orc6 in septin complex function. Orc6 might be a part of a control mechanism directing the cytokinesis machinery during the final steps of mitosis.

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Differential regulation of cellular activities by GTPase-activating protein and NF1.

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2497 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2497-2503 ◽

Cited By ~ 19

Author(s):

N al-Alawi ◽

G Xu ◽

R White ◽

R Clark ◽

F McCormick ◽

...

Keyword(s):

Signal Transduction ◽

Biological Effects ◽

Type I ◽

Type Ii ◽

Gtpase Activity ◽

Ras Proteins ◽

Ras Gtpase ◽

Intrinsic Gtpase Activity

The regulation of the GTPase activity of the Ras proteins is thought to be a key element of signal transduction. Ras proteins have intrinsic GTPase activity and are active in signal transduction when bound to GTP but not following hydrolysis of GTP to GDP. Three cellular Ras GTPase-activating proteins (Ras-gaps) which increase the GTPase activity of wild-type (wt) Ras but not activated Ras in vitro have been identified: type I and type II GAP and type I NF1. Mutations of wt Ras resulting in lowered intrinsic GTPase activity or loss of response to cellular Ras-gap proteins are thought to be the primary reason for the transforming properties of the Ras proteins. In vitro assays show type I and type II GAP and the GAP-related domain of type I NF1 to have similar biochemical properties with respect to activation of the wt Ras GTPase, and it appears as though both type I GAP and NF1 can modulate the GTPase function of Ras in cells. Here we report the assembling of a full-length coding clone for type I NF1 and the biological effects of microinjection of Ras and Ras-gap proteins into fibroblasts. We have found that type I GAP, type II GAP, and type I NF1 show markedly different biological activities in vivo. Coinjection of type I GAP or type I NF1, but not type II GAP, with wt Ras abolished the ability of wt Ras to induce expression from an AP-1-controlled reporter gene. We also found that serum-stimulated DNA synthesis was reduced by prior injection of cells with type I GAP but not type II GAP or type I NF1. These results suggest that type I GAP, type II GAP, and type I NF1 may have different activities in vivo and support the hypothesis that while type I forms of GAP and NF1 may act as negative regulators of wt Ras, they may do so with differential efficiencies.

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