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 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 The substrate specificity of the human TRAPPII complex’s Rab-guanine nucleotide exchange factor activity

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Meredith L. Jenkins ◽  
Noah J. Harris ◽  
Udit Dalwadi ◽  
Kaelin D. Fleming ◽  
Daniel S. Ziemianowicz ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Binding Interface ◽  
Nucleotide Exchange Factor ◽  
Biochemical Characterisation ◽  
Hydrogen Deuterium ◽  
Insight Into

AbstractThe TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.

scholarly journals Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling

2012 ◽  
Vol 23 (14) ◽  
pp. 2723-2740 ◽  
Author(s):  
Steve Jean ◽  
Sarah Cox ◽  
Eric J. Schmidt ◽  
Fred L. Robinson ◽  
Amy Kiger
Keyword(s):  
Endosomal Trafficking ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Phosphatidylinositol 3 Kinase ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Membrane Compartment ◽  
Endosomal Pathway ◽  
Gtpase Activation ◽  
Phosphatidylinositol 3

Cells rely on the coordinated regulation of lipid phosphoinositides and Rab GTPases to define membrane compartment fates along distinct trafficking routes. The family of disease-related myotubularin (MTM) phosphoinositide phosphatases includes catalytically inactive members, or pseudophosphatases, with poorly understood functions. We found that Drosophila MTM pseudophosphatase Sbf coordinates both phosphatidylinositol 3-phosphate (PI(3)P) turnover and Rab21 GTPase activation in an endosomal pathway that controls macrophage remodeling. Sbf dynamically interacts with class II phosphatidylinositol 3-kinase and stably recruits Mtm to promote turnover of a PI(3)P subpool essential for endosomal trafficking. Sbf also functions as a guanine nucleotide exchange factor that promotes Rab21 GTPase activation associated with PI(3)P endosomes. Of importance, Sbf, Mtm, and Rab21 function together, along with Rab11-mediated endosomal trafficking, to control macrophage protrusion formation. This identifies Sbf as a critical coordinator of PI(3)P and Rab21 regulation, which specifies an endosomal pathway and cortical control.

scholarly journals PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72

2019 ◽  
Author(s):  
Sophie Vieweg ◽  
Katie Mulholland ◽  
Bastian Bräuning ◽  
Nitin Kachariya ◽  
Yu-Chiang Lai ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Therapeutic Potential ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Lrrk2 Kinase ◽  
Genetic Code Expansion

AbstractLoss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111-phosphorylated Rab1B that does not demonstrate any major changes suggesting that the phosphorylated SF3 motif may disrupt effector-Switch II interactions. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A suggesting that small molecule activators of PINK1 may have therapeutic potential in patients harbouring LRRK2 mutations.

Identification and characterisation of novel Mss4-binding Rab GTPases

2011 ◽  
Vol 392 (3) ◽  
Author(s):  
Viktor Wixler ◽  
Ludmilla Wixler ◽  
Anika Altenfeld ◽  
Stephan Ludwig ◽  
Roger S. Goody ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mutational Analysis ◽  
Binding Capacity ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Binding Studies ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Abstract The Mss4 (mammalian suppressor of yeast Sec4) is an evolutionarily highly conserved protein and is expressed in all mammalian tissues. Although its precise biological function is still elusive, it has been shown to associate with a subset of secretory Rab proteins (Rab1b, Rab3a, Rab8a, Rab10) and to possess a rather low guanine nucleotide exchange factor (GEF) activity towards them in vitro (Rab1, Rab3a and Rab8a). By screening a human placenta cDNA library with Mss4 as bait, we identified several Rab GTPases (Rab12, Rab13 and Rab18) as novel Mss4-binding Rab proteins. Only exocytic but no endocytic Rab GTPases were found in our search. The binding of Mss4 to Rab proteins was confirmed by direct yeast two-hybrid interaction, by co-immunoprecipitation from lysates of mammalian cells, by immunofluorescence colocalisation as well as by direct in vitro binding studies. Analysis of Mss4 catalytic activity towards different Rab substrates confirmed that it is a somewhat inefficient GEF. These data, together with our mutational analysis of Mss4-Rab binding capacity, support the already proposed idea that Mss4 functions rather as a chaperone for exocytic Rab GTPases than as a GEF.

scholarly journals Rabex-5 Is a Rab22 Effector and Mediates a Rab22-Rab5 Signaling Cascade in Endocytosis

2009 ◽  
Vol 20 (22) ◽  
pp. 4720-4729 ◽  
Author(s):  
Huaiping Zhu ◽  
Zhimin Liang ◽  
Guangpu Li
Keyword(s):  
Membrane Targeting ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Hairpin Rna ◽  
Guanosine Diphosphate ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Early Endosomes ◽  
Epidermal Growth

Rabex-5 targets to early endosomes and functions as a guanine nucleotide exchange factor for Rab5. Membrane targeting is critical for Rabex-5 to activate Rab5 on early endosomes in the cell. Here, we report the identification of Rab22 as a binding site on early endosomes for direct recruitment of Rabex-5 and activation of Rab5, establishing a Rab22-Rab5 signaling relay to promote early endosome fusion. Rab22 in guanosine 5′-O-(3-thio)triphosphate-loaded form, but not guanosine diphosphate-loaded form, binds to the early endosomal targeting domain (residues 81-230) of Rabex-5 in pull-down assays. Rabex-5 targets to Rab22-containing early endosomes, and Rab22 knockdown by short hairpin RNA abrogates the membrane targeting of Rabex-5 in the cell. In addition, coexpression of Rab22 and Rab5 shows synergistic enlargement of early endosomes, and this synergy is dependent on Rabex-5, providing further support for the collaboration of the two Rab GTPases in regulation of endosome dynamics. This novel Rab22–Rabex-5–Rab5 cascade is functionally important for the endocytosis and degradation of epidermal growth factor.

scholarly journals C9orf72, a protein associated with amyotrophic lateral sclerosis (ALS) is a guanine nucleotide exchange factor

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5815 ◽  
Author(s):  
Shalini Iyer ◽  
Vasanta Subramanian ◽  
K. Ravi Acharya
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Guanine Nucleotide Exchange Factor ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Mtorc1 Signaling ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two late onset neurodegenerative diseases, have been shown to share overlapping cellular pathologies and genetic origins. Studies suggest that a hexanucleotide repeat expansion in the first intron of the C9orf72 gene is the most common cause of familial FTD and ALS pathology. The C9orf72 protein is predicted to be a differentially expressed in normal and neoplastic cells domain protein implying that C9orf72 functions as a guanine nucleotide exchange factor (GEF) to regulate specific Rab GTPases. Reported studies thus far point to a putative role for C9orf72 in lysosome biogenesis, vesicular trafficking, autophagy and mechanistic target of rapamycin complex1 (mTORC1) signaling. Here we report the expression, purification and biochemical characterization of C9orf72 protein. We conclusively show that C9orf72 is a GEF. The distinctive presence of both Rab- and Rho-GTPase GEF activities suggests that C9orf72 may function as a dual exchange factor coupling physiological functions such as cytoskeleton modulation and autophagy with endocytosis.

scholarly journals A Gs-RhoGEF interaction: An old G protein finds a new job

2020 ◽  
Vol 295 (50) ◽  
pp. 16929-16930
Author(s):  
Vladlen Z. Slepak ◽  
Alexey Pronin
Keyword(s):  
G Protein ◽  
Rho Gtpases ◽  
Small Gtpases ◽  
Adenylyl Cyclases ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Downstream Effectors ◽  
Cytoskeletal Rearrangements

The heterotrimeric G proteins are known to have a variety of downstream effectors, but Gs was long thought to be specifically coupled to adenylyl cyclases. A new study indicates that activated Gs can also directly interact with a guanine nucleotide exchange factor for Rho family small GTPases, PDZ-RhoGEF. This novel interaction mediates activation of the small G protein Cdc42 by Gs-coupled GPCRs, inducing cytoskeletal rearrangements and formation of filopodia-like structures. Furthermore, overexpression of a minimal PDZ-RhoGEF fragment can down-regulate cAMP signaling, suggesting that this effector competes with canonical signaling. This first demonstration that the Gαs subfamily regulates activity of Rho GTPases extends our understanding of Gαs activity and establishes RhoGEF coupling as a universal Gα function.

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.

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