scholarly journals GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

1998 ◽  
Vol 18 (2) ◽  
pp. 827-838 ◽  
Author(s):  
Celeste J. Richardson ◽  
Sara Jones ◽  
Robert J. Litt ◽  
Nava Segev
Keyword(s):  
Membrane Fusion ◽  
Vesicular Transport ◽  
Gtpase Activity ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Dominant Phenotype ◽  
Guanine Nucleotide Exchange ◽  
Membrane Morphology ◽  
Nucleotide Exchange Factor ◽  
Mutant Cells

ABSTRACT GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37°C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-freeYPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.

scholarly journals Eng2, a new player involved in feedback loop regulation of Cdc42 activity in fission yeast

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Patricia García ◽  
Pedro M. Coll ◽  
Francisco del Rey ◽  
M. Isabel Geli ◽  
Pilar Pérez ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Feedback Loop ◽  
Oscillatory Behavior ◽  
Small Gtpase ◽  
Dual Function ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Endocytic Machinery ◽  
Mutant Cells

AbstractCell polarity and morphogenesis are regulated by the small GTPase Cdc42. Even though major advances have been done in the field during the last years, the molecular details leading to its activation in particular cellular contexts are not completely understood. In fission yeast, the β(1,3)-glucanase Eng2 is a “moonlighting protein” with a dual function, acting as a hydrolase during spore dehiscence, and as component of the endocytic machinery in vegetative cells. Here, we report that Eng2 plays a role in Cdc42 activation during polarized growth through its interaction with the scaffold protein Scd2, which brings Cdc42 together with its guanine nucleotide exchange factor (GEF) Scd1. eng2Δ mutant cells have defects in activation of the bipolar growth (NETO), remaining monopolar during all the cell cycle. In the absence of Eng2 the accumulation of Scd1 and Scd2 at the poles is reduced, the levels of Cdc42 activation decrease, and the Cdc42 oscillatory behavior, associated with bipolar growth in wild type cells, is altered. Furthermore, overexpression of Eng2 partially rescues the growth and polarity defects of a cdc42-L160S mutant. Altogether, our work unveils a new factor regulating the activity of Cdc42, which could potentially link the polarity and endocytic machineries.

scholarly journals A Vps21 endocytic module regulates autophagy

2014 ◽  
Vol 25 (20) ◽  
pp. 3166-3177 ◽  
Author(s):  
Yong Chen ◽  
Fan Zhou ◽  
Shenshen Zou ◽  
Sidney Yu ◽  
Shaoshan Li ◽  
...  
Keyword(s):  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Downstream Effectors ◽  
Fluorescence And Electron Microscopy ◽  
Late Endosomes ◽  
Cellular Components ◽  
Mutant Cells ◽  
Additional Role

In autophagy, the double-membrane autophagosome delivers cellular components for their degradation in the lysosome. The conserved Ypt/Rab GTPases regulate all cellular trafficking pathways, including autophagy. These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors. Rab7 and its yeast homologue, Ypt7, in the context of such a module, regulate the fusion of both late endosomes and autophagosomes with the lysosome. In yeast, the Rab5-related Vps21 is known for its role in early- to late-endosome transport. Here we show an additional role for Vps21 in autophagy. First, vps21∆ mutant cells are defective in selective and nonselective autophagy. Second, fluorescence and electron microscopy analyses show that vps21∆ mutant cells accumulate clusters of autophagosomal structures outside the vacuole. Third, cells with mutations in other members of the endocytic Vps21 module, including the GEF Vps9 and factors that function downstream of Vps21, Vac1, CORVET, Pep12, and Vps45, are also defective in autophagy and accumulate clusters of autophagosomes. Finally, Vps21 localizes to PAS. We propose that the endocytic Vps21 module also regulates autophagy. These findings support the idea that the two pathways leading to the lysosome—endocytosis and autophagy—converge through the Vps21 and Ypt7 GTPase modules.

scholarly journals Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases determined in parallel

2019 ◽  
Vol 294 (25) ◽  
pp. 9937-9948 ◽  
Author(s):  
Ryan C. Killoran ◽  
Matthew J. Smith
Keyword(s):  
Binding Pocket ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Activation Potential ◽  
Single Complex ◽  
Ras Isoforms

Small GTPases alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly the three RAS isoforms HRAS, KRAS, and NRAS and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is some evidence of direct biochemical cross-talk between their functional G-domains. The activation potential of a given GTPase is contingent on a codependent interaction with the nucleotide and a Mg2+ ion, which bind to individual variants with distinct affinities coordinated by residues in the GTPase nucleotide-binding pocket. Here, we utilized a selective-labeling strategy coupled with real-time NMR spectroscopy to monitor nucleotide exchange, GTP hydrolysis, and effector interactions of multiple small GTPases in a single complex system. We provide insight into nucleotide preference and the role of Mg2+ in activating both WT and oncogenic mutant enzymes. Multiplexing revealed guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP), and effector-binding specificities in mixtures of GTPases and resolved that the three related RAS isoforms are biochemically equivalent. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately determine an activation potential for any given GTPase, as small GTPases such as RAS-like proto-oncogene A (RALA) or the G12C mutant of KRAS display fast exchange kinetics but have a high affinity for GDP. Furthermore, we propose that the G-domains of small GTPases behave autonomously in solution and that nucleotide cycling proceeds independently of protein concentration but is highly impacted by Mg2+ abundance.

scholarly journals The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2

2020 ◽  
Vol 295 (34) ◽  
pp. 12130-12142 ◽  
Author(s):  
Megan E. Arrington ◽  
Brenda Temple ◽  
Antje Schaefer ◽  
Sharon L. Campbell
Keyword(s):  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Ras Gtpases ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Parent Protein ◽  
Activating Mutation ◽  
P21 Activated Kinase ◽  
Effector Pathways ◽  
Effector Binding

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K. Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K. As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

scholarly journals The Role of Trs65 in the Ypt/Rab Guanine Nucleotide Exchange Factor Function of the TRAPP II Complex

2007 ◽  
Vol 18 (7) ◽  
pp. 2533-2541 ◽  
Author(s):  
Yongheng Liang ◽  
Nadya Morozova ◽  
Andrei A. Tokarev ◽  
Jonathan W. Mulholland ◽  
Nava Segev
Keyword(s):  
Intracellular Trafficking ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Cell Wall Biogenesis ◽  
Nucleotide Exchange Factor ◽  
Low Levels ◽  
Mutant Cells ◽  
Factor Function

The conserved modular complex TRAPP is a guanine nucleotide exchanger (GEF) for the yeast Golgi Ypt-GTPase gatekeepers. TRAPP I and TRAPP II share seven subunits and act as GEFs for Ypt1 and Ypt31/32, respectively, which in turn regulate transport into and out of the Golgi. Trs65/Kre11 is one of three TRAPP II-specific subunits. Unlike the other two subunits, Trs120 and Trs130, Trs65 is not essential for viability, is conserved only among some fungi, and its contribution to TRAPP II function is unclear. Here, we provide genetic, biochemical, and cellular evidence for the role of Trs65 in TRAPP II function. First, like Trs130, Trs65 localizes to the trans-Golgi. Second, TRS65 interacts genetically with TRS120 and TRS130. Third, Trs65 interacts physically with Trs120 and Trs130. Finally, trs65 mutant cells have low levels of Trs130 protein, and they are defective in the GEF activity of TRAPP II and the intracellular distribution of Ypt1 and Ypt31/32. Together, these results show that Trs65 plays a role in the Ypt GEF activity of TRAPP II in concert with the two other TRAPP II-specific subunits. Elucidation of the role played by Trs65 in intracellular trafficking is important for understanding how this process is coordinated with two other processes in which Trs65 is implicated: cell wall biogenesis and stress response.

scholarly journals Conserved NDR/LATS kinase controls RAS GTPase activity to regulate cell growth and chronological lifespan

2019 ◽  
Vol 30 (20) ◽  
pp. 2598-2616 ◽  
Author(s):  
Chuan Chen ◽  
Marbelys Rodriguez Pino ◽  
Patrick Roman Haller ◽  
Fulvia Verde
Keyword(s):  
Cell Growth ◽  
Gtpase Activity ◽  
Nucleotide Exchange ◽  
Chronological Lifespan ◽  
Protein Levels ◽  
Ras Gtpase ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Regulate Cell Growth ◽  
Cell Growth And Differentiation

Adaptation to the nutritional environment is critical for all cells. RAS GTPase is a highly conserved GTP-binding protein with crucial functions for cell growth and differentiation in response to environmental conditions. Here, we describe a novel mechanism connecting RAS GTPase to nutrient availability in fission yeast. We report that the conserved NDR/LATS kinase Orb6 responds to nutritional cues and regulates Ras1 GTPase activity. Orb6 increases the protein levels of an Ras1 GTPase activator, the guanine nucleotide exchange factor Efc25, by phosphorylating Sts5, a protein bound to efc25 mRNA. By manipulating the extent of Orb6-mediated Sts5 assembly into RNP granules, we can modulate Efc25 protein levels, Ras1 GTPase activity, and, as a result, cell growth and cell survival. Thus, we conclude that the Orb6–Sts5–Ras1 regulatory axis plays a crucial role in promoting cell adaptation, balancing the opposing demands of promoting cell growth and extending chronological lifespan.

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