scholarly journals Septin ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p

2002 ◽  
Vol 156 (2) ◽  
pp. 315-326 ◽  
Author(s):  
Amy S. Gladfelter ◽  
Indrani Bose ◽  
Trevin R. Zyla ◽  
Elaine S.G. Bardes ◽  
Daniel J. Lew
Keyword(s):  
Transcriptional Activation ◽  
Gtpase Activating Protein ◽  
Gtp Hydrolysis ◽  
Septin Ring ◽  
Turn On ◽  
Ring Assembly ◽  
Bud Emergence ◽  
Assembly Factor ◽  
Gtpase Cycle ◽  
Budding Yeast Cell Cycle

At the beginning of the budding yeast cell cycle, the GTPase Cdc42p promotes the assembly of a ring of septins at the site of future bud emergence. Here, we present an analysis of cdc42 mutants that display specific defects in septin organization, which identifies an important role for GTP hydrolysis by Cdc42p in the assembly of the septin ring. The mutants show defects in basal or stimulated GTP hydrolysis, and the septin misorganization is suppressed by overexpression of a Cdc42p GTPase-activating protein (GAP). Other mutants known to affect GTP hydrolysis by Cdc42p also caused septin misorganization, as did deletion of Cdc42p GAPs. In performing its roles in actin polarization and transcriptional activation, GTP-Cdc42p is thought to function by activating and/or recruiting effectors to the site of polarization. Excess accumulation of GTP-Cdc42p due to a defect in GTP hydrolysis by the septin-specific alleles might cause unphysiological activation of effectors, interfering with septin assembly. However, the recessive and dose-sensitive genetic behavior of the septin-specific cdc42 mutants is inconsistent with the septin defect stemming from a dominant interference of this type. Instead, we suggest that assembly of the septin ring involves repeated cycles of GTP loading and GTP hydrolysis by Cdc42p. These results suggest that a single GTPase, Cdc42p, can act either as a ras-like GTP-dependent “switch” to turn on effectors or as an EF-Tu–like “assembly factor” using the GTPase cycle to assemble a macromolecular structure.

scholarly journals Dynamics of septin ring and collar formation in Saccharomyces cerevisiae

2011 ◽  
Vol 392 (8-9) ◽  
pp. 689-697 ◽  
Author(s):  
Hsin Chen ◽  
Audrey S. Howell ◽  
Alex Robeson ◽  
Daniel J. Lew
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Septin Ring ◽  
Ring Assembly ◽  
Bud Emergence ◽  
Steady Rate ◽  
Constant Rate ◽  
Bud Neck ◽  
Quantitative Analyses ◽  
Reduced Rate ◽  
Mother Cells

Abstract Although the septin ring and collar in budding yeast were described over 20 years ago, there is still controversy regarding the organization of septin filaments within these structures and about the way in which the ring first forms and about how it converts into a collar at the mother-bud neck. Here we present quantitative analyses of the recruitment of fluorescently-tagged septins to the ring and collar through the cell cycle. Septin ring assembly began several minutes after polarity establishment and this interval was longer in daughter than in mother cells, suggesting asymmetric inheritance of septin regulators. Septins formed an initial faint and irregular ring, which became more regular as septins were recruited at a constant rate. This steady rate of septin recruitment continued for several minutes after the ring converted to a collar at bud emergence. We did not detect a stepwise change in septin fluorescence during the ring-to-collar transition. After collar formation, septins continued to accumulate at the bud neck, though at a reduced rate, until the onset of cytokinesis when the amount of neck-localized septins rapidly decreased. Implications for the mechanism of septin ring assembly are discussed.

Membrane curvature and the control of GTP hydrolysis in Arf1 during COPI vesicle formation

2005 ◽  
Vol 33 (4) ◽  
pp. 619-622 ◽  
Author(s):  
B. Antonny ◽  
J. Bigay ◽  
J.-F. Casella ◽  
G. Drin ◽  
B. Mesmin ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Membrane Curvature ◽  
Gtp Hydrolysis ◽  
Membrane Fission ◽  
Transport Vesicles ◽  
Highly Sensitive ◽  
Copi Vesicle ◽  
Membrane Budding ◽  
Gtpase Cycle ◽  
Adp Ribosylation Factor

The GTP switch of the small G-protein Arf1 (ADP-ribosylation factor 1) on lipid membranes promotes the polymerization of the COPI (coat protein complex I) coat, which acts as a membrane deforming shell to form transport vesicles. Real-time measurements for coat assembly on liposomes gives insights into how the GTPase cycle of Arf1 is coupled in time with the polymerization of the COPI coat and the resulting membrane deformation. One key parameter seems to be the membrane curvature. Arf-GAP1 (where GAP stands for GTPase-activating protein), which promotes GTP hydrolysis in the Arf1–COPI complex is highly sensitive to lipid packing. Its activity on Arf1-GTP increases by two orders of magnitude as the diameter of the liposomes approaches that of authentic transport vesicles (60 nm). This suggests that during membrane budding, Arf1-GTP molecules are progressively eliminated from the coated area where the membrane curvature is positive, but are protected from Arf-GAP1 at the bud neck due to the negative curvature of this region. As a result, the coat should be stable as long as the bud remains attached and should disassemble as soon as membrane fission occurs.

scholarly journals Structural consequences of the interaction of RbgA with a 50S ribosomal subunit assembly intermediate

2019 ◽  
Vol 47 (19) ◽  
pp. 10414-10425 ◽  
Author(s):  
Amal Seffouh ◽  
Nikhil Jain ◽  
Dushyant Jahagirdar ◽  
Kaustuv Basu ◽  
Aida Razi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Catalytic Residue ◽  
Gtp Hydrolysis ◽  
Subunit Assembly ◽  
Cryo Electron Microscopy ◽  
Assembly Factor ◽  
Assembly Intermediate ◽  
Immature Particle

Abstract Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.

scholarly journals Coordination of the leucine-sensing Rag GTPase cycle by leucyl-tRNA synthetase in the mTORC1 signaling pathway

2018 ◽  
Vol 115 (23) ◽  
pp. E5279-E5288 ◽  
Author(s):  
Minji Lee ◽  
Jong Hyun Kim ◽  
Ina Yoon ◽  
Chulho Lee ◽  
Mohammad Fallahi Sichani ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Trna Synthetase ◽  
Gtp Hydrolysis ◽  
Mtorc1 Signaling ◽  
Amino Acid Sensing ◽  
Gtpase Cycle ◽  
Rag Gtpase ◽  
Cancer Tissues ◽  
Mtorc1 Activation ◽  
Hydrolysis Of

A protein synthesis enzyme, leucyl-tRNA synthetase (LRS), serves as a leucine sensor for the mechanistic target of rapamycin complex 1 (mTORC1), which is a central effector for protein synthesis, metabolism, autophagy, and cell growth. However, its significance in mTORC1 signaling and cancer growth and its functional relationship with other suggested leucine signal mediators are not well-understood. Here we show the kinetics of the Rag GTPase cycle during leucine signaling and that LRS serves as an initiating “ON” switch via GTP hydrolysis of RagD that drives the entire Rag GTPase cycle, whereas Sestrin2 functions as an “OFF” switch by controlling GTP hydrolysis of RagB in the Rag GTPase–mTORC1 axis. The LRS–RagD axis showed a positive correlation with mTORC1 activity in cancer tissues and cells. The GTP–GDP cycle of the RagD–RagB pair, rather than the RagC–RagA pair, is critical for leucine-induced mTORC1 activation. The active RagD–RagB pair can overcome the absence of the RagC–RagA pair, but the opposite is not the case. This work suggests that the GTPase cycle of RagD–RagB coordinated by LRS and Sestrin2 is critical for controlling mTORC1 activation, and thus will extend the current understanding of the amino acid-sensing mechanism.

scholarly journals Differential GAP requirement for Cdc42-GTP polarization during proliferation and sexual reproduction

2018 ◽  
Vol 217 (12) ◽  
pp. 4215-4229 ◽  
Author(s):  
Daniela Gallo Castro ◽  
Sophie G. Martin
Keyword(s):  
Sexual Reproduction ◽  
Cell Types ◽  
Cell Polarization ◽  
Dynamic Polarization ◽  
Gtpase Activating Protein ◽  
Triple Mutant ◽  
Local Zone ◽  
Gtp Hydrolysis ◽  
Extrinsic Cues ◽  
Mutant Cells

The formation of a local zone of Cdc42 GTPase activity, which governs cell polarization in many cell types, requires not only local activation but also switch-off mechanisms. In this study, we identify Rga3, a paralog of Rga4, as a novel Cdc42 GTPase-activating protein (GAP) in the fission yeast Schizosaccharomyces pombe. Contrary to Rga4, Rga3 localizes with Cdc42-GTP to sites of polarity. Rga3 is dispensable for cell polarization during mitotic growth, but it limits the lifetime of unstable Cdc42-GTP patches that underlie cell pairing during sexual reproduction, masking a partly compensatory patch-wandering motion. In consequence, cells lacking rga3 hyperpolarize and lose out in mating competition. Rga3 synergizes with the Cdc42 GAPs Rga4 and Rga6 to restrict Cdc42-GTP zone sizes during mitotic growth. Surprisingly, triple-mutant cells, which are almost fully round, retain pheromone-dependent dynamic polarization of Cdc42-GTP, extend a polarized projection, and mate. Thus, the requirement for Cdc42-GTP hydrolysis by GAPs is distinct during polarization by intrinsic or extrinsic cues.

scholarly journals Ribosome assembly factor URB1 contributes to colorectal cancer proliferation through transcriptional activation of ATF4

2020 ◽  
Vol 112 (1) ◽  
pp. 101-116
Author(s):  
Tao Wang ◽  
Lai‐Yuan Li ◽  
Yi‐Feng Chen ◽  
Si‐Wu Fu ◽  
Zhi‐Wei Wu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Transcriptional Activation ◽  
Ribosome Assembly ◽  
Cancer Proliferation ◽  
Assembly Factor

scholarly journals Oligomerization and Dissociation of AP-1 Adaptors Are Regulated by Cargo Signals and by ArfGAP1-induced GTP Hydrolysis

2005 ◽  
Vol 16 (10) ◽  
pp. 4745-4754 ◽  
Author(s):  
Daniel M. Meyer ◽  
Pascal Crottet ◽  
Bohumil Maco ◽  
Elena Degtyar ◽  
Dan Cassel ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Adaptor Proteins ◽  
Gtpase Activity ◽  
Gtpase Activating Protein ◽  
Gtp Hydrolysis ◽  
Sorting Signals ◽  
Initial Assembly ◽  
Cargo Sorting

The mechanism of AP-1/clathrin coat formation was analyzed using purified adaptor proteins and synthetic liposomes presenting tyrosine sorting signals. AP-1 adaptors recruited in the presence of Arf1·GTP and sorting signals were found to oligomerize to high-molecular-weight complexes even in the absence of clathrin. The appendage domains of the AP-1 adaptins were not required for oligomerization. On GTP hydrolysis induced by the GTPase-activating protein ArfGAP1, the complexes were disassembled and AP-1 dissociated from the membrane. AP-1 stimulated ArfGAP1 activity, suggesting a role of AP-1 in the regulation of the Arf1 “GTPase timer.” In the presence of cytosol, AP-1 could be recruited to liposomes without sorting signals, consistent with the existence of docking factors in the cytosol. Under these conditions, however, AP-1 remained monomeric, and recruitment in the presence of GTP was short-lived. Sorting signals allowed stable recruitment and oligomerization also in the presence of cytosol. These results suggest a mechanism whereby initial assembly of AP-1 with Arf1·GTP and ArfGAP1 on the membrane stimulates Arf1 GTPase activity, whereas interaction with cargo induces oligomerization and reduces the rate of GTP hydrolysis, thus contributing to efficient cargo sorting.

scholarly journals Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release

1992 ◽  
Vol 287 (2) ◽  
pp. 555-559 ◽  
Author(s):  
M R Webb ◽  
J L Hunter
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Protein A ◽  
Gtpase Activity ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Gtp Hydrolysis ◽  
Type Protein ◽  
Wild Type Protein ◽  
Kinetics Of ◽  
Continuous Assay

The mechanism of GTPase-activating protein (GAP) activation of p21ras GTP hydrolysis has been investigated by measuring the kinetics of release of Pi during the hydrolysis. The measurement uses a continuous spectroscopic assay for Pi, based on a guanosine analogue, 2-amino-6-mercapto-7-methylpurine ribonucleoside, as substrate for purine nucleoside phosphorylase [Webb, M.R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4884-4887]. This phosphorolysis gives an absorbance increase at 360 nm, so that when the reaction is coupled to GTP hydrolysis, the change in absorbance gives the total amount of Pi released from the p21ras. The rate of the absorbance increase gives the GTPase activity. This provides a non-radioactive method of determining p21ras concentration and GAP activity. It was used to determine the interaction of GAP with wild-type p21ras and two mutants (Leu-61/Ser-186 and Asp-12), all in the GTP (or guanosine 5′-[beta gamma-imido]triphosphate) form. The Leu-61/Ser-186 mutant binds 10-fold tighter than does the wild-type protein. The Asp-12 mutant binds to GAP with the same affinity as the wild-type protein. A novel GTPase activity was characterized whereby the EDTA-induced nucleotide release and GAP-activated cleavage of bound GTP leads to steady-state turnover of GTP hydrolysis. An assay for GAP is described based on this activity.

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