scholarly journals The small GTPase Rab5a and its guanine nucleotide exchange factors are involved in post-Golgi trafficking of storage proteins in developing soybean cotyledon

2019 ◽  
Vol 71 (3) ◽  
pp. 808-822 ◽  
Author(s):  
Zhongyan Wei ◽  
Tian Pan ◽  
Yuyang Zhao ◽  
Bohong Su ◽  
Yulong Ren ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Storage Protein ◽  
Storage Proteins ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Protein Storage ◽  
Soybean Cotyledon ◽  
Guanine Nucleotide Exchange ◽  
Protein Storage Vacuoles

Abstract Storage protein is the most abundant nutritional component in soybean seed. Morphology-based evidence has verified that storage proteins are initially synthesized on the endoplasmic reticulum, and then follow the Golgi-mediated pathway to the protein storage vacuole. However, the molecular mechanisms of storage protein trafficking in soybean remain unknown. Here, we clone the soybean homologs of Rab5 and its guanine nucleotide exchange factor (GEF) VPS9. GEF activity combined with yeast two-hybrid assays demonstrated that GmVPS9a2 might specifically act as the GEF of the canonical Rab5, while GmVPS9b functions as a common activator for all Rab5s. Subcellular localization experiments showed that GmRab5a was dually localized to the trans-Golgi network and pre-vacuolar compartments in developing soybean cotyledon cells. Expression of a dominant negative variant of Rab5a, or RNAi of either Rab5a or GmVPS9s, significantly disrupted trafficking of mRFP–CT10, a cargo marker for storage protein sorting, to protein storage vacuoles in maturing soybean cotyledons. Together, our results systematically revealed the important role of GmRab5a and its GEFs in storage protein trafficking, and verified the transient expression system as an efficient approach for elucidating storage protein trafficking mechanisms in seed.

scholarly journals Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

2018 ◽  
Author(s):  
Meng Shi ◽  
Bing Chen ◽  
Boon Kim Boh ◽  
Yan Zhou ◽  
Divyanshu Mahajan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Retrograde Trafficking ◽  
Guanine Nucleotide Exchange ◽  
Trafficking Pathway ◽  
Nucleotide Exchange Factor ◽  
Mtorc1 Signaling ◽  
Nutrient Signaling

AbstractThe endosome-to-Golgi or endocytic retrograde trafficking pathway is an important post-Golgi recycling route. We made a novel discovery that the retrograde trafficking of cargos is inhibited and stimulated by the absence and presence, respectively, of amino acids (AAs), especially glutamine. By testing components of the AA-stimulated mTORC1 signaling pathway, we demonstrated that SLC38A9, v-ATPase and Ragulator, but not Rag GTPases and mTORC1, are essential for the AA-stimulated trafficking. Arl5, an ARF-like family small GTPase, interacts with Ragulator in an AA-regulated manner and both Arl5 and its effector, the Golgi-associated retrograde protein complex (GARP), are required for the AA-stimulated trafficking. We have therefore identified a mechanistic connection between the nutrient signaling and the retrograde trafficking pathway, whereby SLC38A9 and v-ATPase sense AA-sufficiency and Ragulator functions as a guanine nucleotide exchange factor to activate Arl5, which, together with GARP, a tethering factor, probably facilitates the endosome-to-Golgi trafficking.

scholarly journals G1/S Cyclin-dependent Kinase Regulates Small GTPase Rho1p through Phosphorylation of RhoGEF Tus1p inSaccharomyces cerevisiae

2008 ◽  
Vol 19 (4) ◽  
pp. 1763-1771 ◽  
Author(s):  
Keiko Kono ◽  
Satoru Nogami ◽  
Mitsuhiro Abe ◽  
Masafumi Nishizawa ◽  
Shinichi Morishita ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Cyclin Dependent Kinase ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Temporal And Spatial ◽  
Cdk Phosphorylation

Rho1p is an essential small GTPase that plays a key role in the morphogenesis of Saccharomyces cerevisiae. We show here that the activation of Rho1p is regulated by a cyclin-dependent kinase (CDK). Rho1p is activated at the G1/S transition at the incipient-bud sites by the Cln2p (G1 cyclin) and Cdc28p (CDK) complex, in a process mediated by Tus1p, a guanine nucleotide exchange factor for Rho1p. Tus1p interacts physically with Cln2p/Cdc28p and is phosphorylated in a Cln2p/Cdc28p-dependent manner. CDK phosphorylation consensus sites in Tus1p are required for both Cln2p-dependent activation of Rho1p and polarized organization of the actin cytoskeleton. We propose that Cln2p/Cdc28p-dependent phosphorylation of Tus1p is required for appropriate temporal and spatial activation of Rho1p at the G1/S transition.

scholarly journals Ras Binding Triggers Ubiquitination of the Ras Exchange Factor Ras-GRF2

2001 ◽  
Vol 21 (6) ◽  
pp. 2107-2117 ◽  
Author(s):  
Carmen L. de Hoog ◽  
Jackie A. Koehler ◽  
Marni D. Goldstein ◽  
Paul Taylor ◽  
Daniel Figeys ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Small Gtpase ◽  
Dominant Negative ◽  
26S Proteasome ◽  
Guanine Nucleotide ◽  
Sequence Motifs ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange

ABSTRACT Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.

scholarly journals Divergent Mechanisms Activating RAS and Small GTPases Through Post-translational Modification

2021 ◽  
Vol 8 ◽  
Author(s):  
Natsuki Osaka ◽  
Yoshihisa Hirota ◽  
Doshun Ito ◽  
Yoshiki Ikeda ◽  
Ryo Kamata ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Guanine Nucleotide Exchange ◽  
Ras Superfamily ◽  
Switch Regions

RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound “OFF” state and GTP-bound “ON” state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound “ON” state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.

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