Measuring Rab GTPase-Activating Protein (GAP) Activity in Live Cells and Extracts

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.

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Identification of a Rab GTPase-activating protein cascade that controls recycling of the Rab5 GTPase Vps21 from the vacuole

Molecular Biology of the Cell ◽

10.1091/mbc.e15-02-0062 ◽

2015 ◽

Vol 26 (13) ◽

pp. 2535-2549 ◽

Cited By ~ 21

Author(s):

Meenakshi Rana ◽

Jens Lachmann ◽

Christian Ungermann

Keyword(s):

Endoplasmic Reticulum ◽

Endocytic Pathway ◽

Rab Gtpase ◽

Guanine Nucleotide ◽

Membrane Recycling ◽

Nucleotide Exchange ◽

Gtpase Activating Protein ◽

Guanine Nucleotide Exchange ◽

Vacuole Fusion ◽

Nucleotide Exchange Factor

Transport within the endocytic pathway depends on a consecutive function of the endosomal Rab5 and the late endosomal/lysosomal Rab7 GTPases to promote membrane recycling and fusion in the context of endosomal maturation. We previously identified the hexameric BLOC-1 complex as an effector of the yeast Rab5 Vps21, which also recruits the GTPase-activating protein (GAP) Msb3. This raises the question of when Vps21 is inactivated on endosomes. We provide evidence for a Rab cascade in which activation of the Rab7 homologue Ypt7 triggers inactivation of Vps21. We find that the guanine nucleotide exchange factor (GEF) of Ypt7 (the Mon1-Ccz1 complex) and BLOC-1 both localize to the same endosomes. Overexpression of Mon1-Ccz1, which generates additional Ypt7-GTP, or overexpression of activated Ypt7 promotes relocalization of Vps21 from endosomes to the endoplasmic reticulum (ER), which is indicative of Vps21 inactivation. This ER relocalization is prevented by loss of either BLOC-1 or Msb3, but it also occurs in mutants lacking endosome–vacuole fusion machinery such as the HOPS tethering complex, an effector of Ypt7. Importantly, BLOC-1 interacts with the HOPS on vacuoles, suggesting a direct Ypt7-dependent cross-talk. These data indicate that efficient Vps21 recycling requires both Ypt7 and endosome–vacuole fusion, thus suggesting extended control of a GAP cascade beyond Rab interactions.

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Identification of Rab GTPase-Activating Protein-Like Protein (RabGAPLP) as a Novel Alix/AIP1-Interacting Protein

Bioscience Biotechnology and Biochemistry ◽

10.1271/bbb.69.861 ◽

2005 ◽

Vol 69 (4) ◽

pp. 861-865 ◽

Cited By ~ 8

Author(s):

Fumitaka ICHIOKA ◽

Mio HORII ◽

Keiichi KATOH ◽

Yoshinori TERASAWA ◽

Hideki SHIBATA ◽

...

Keyword(s):

Rab Gtpase ◽

Gtpase Activating Protein ◽

Interacting Protein

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Inhibition of GLUT4 Translocation by Tbc1d1, a Rab GTPase-activating Protein Abundant in Skeletal Muscle, Is Partially Relieved by AMP-activated Protein Kinase Activation

Journal of Biological Chemistry ◽

10.1074/jbc.m708934200 ◽

2008 ◽

Vol 283 (14) ◽

pp. 9187-9195 ◽

Cited By ~ 100

Author(s):

Jose A. Chavez ◽

William G. Roach ◽

Susanna R. Keller ◽

William S. Lane ◽

Gustav E. Lienhard

Keyword(s):

Skeletal Muscle ◽

Protein Kinase ◽

Rab Gtpase ◽

Glut4 Translocation ◽

Gtpase Activating Protein ◽

Kinase Activation ◽

Protein Kinase Activation ◽

Amp Activated Protein Kinase

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The GTPase-Activating Protein FgGyp1 Is Important for Vegetative Growth, Conidiation, and Virulence and Negatively Regulates DON Biosynthesis in Fusarium graminearium

Frontiers in Microbiology ◽

10.3389/fmicb.2021.621519 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qiaojia Zheng ◽

Zhi Yu ◽

Yanping Yuan ◽

Danli Sun ◽

Yakubu Saddeeq Abubakar ◽

...

Keyword(s):

Vegetative Growth ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Negative Regulator ◽

Rab Gtpase ◽

Wild Type ◽

Gtpase Activating Protein ◽

Replacement Method

Ypt1 is a small Rab GTPase in yeast, Gyp1 functions at the Golgi as a negative regulator of Ypt1. Gyp1 homologs are conserved in filamentous fungi. However, the roles of Gyp1 in phytopathogenic fungi are still unclear. Herein, we investigated the functions of FgGyp1 in the wheat pathogen Fusarium graminearum by live-cell imaging, genetic, and pathological analyses. Targeted gene replacement method was used to delete FgGYP1 in F. graminearum. Phenotypic analyses showed that FgGyp1 is critically important not only for the vegetative growth of F. graminearum but also its conidiation. The mutant’s vegetative growth was significantly reduced by 70% compared to the wild type PH-1. The virulence of FgGYP1 deletion mutant was significantly decreased when compared with the wild type PH-1. We further found that FgGyp1 negatively regulates DON production of the fungus. Live-cell imaging clearly demonstrated that FgGyp1 mainly localizes to the Golgi apparatus. Moreover, the TBC domain, C-terminal, and N-terminal regions of FgGyp1 are found to be indispensable for its biological functions and normal localization. The Arg357 residue of FgGyp1 is essential for its functions but dispensable for the normal localization of the protein, while the Arg284 residue is not required for both the functions and normal localization of the protein. Furthermore, we showed that FgGyp1 essentially hydrolyzes the GTP-bound FgRab1 (activated form) to its corresponding GDP-bound (inactive) form in vitro, suggesting that FgGyp1 is a GTPase-activating protein (GAP) for FgRab1. Finally, FgGyp1 was found to be important for FgSnc1-mediated fusion of secretory vesicles from the Golgi with the plasma membrane in F. graminearum. Put together, these data demonstrate that FgGyp1 functions as a GAP for FgRab1 and is important for vegetative growth, conidiation and virulence, and negatively regulates DON biosynthesis in F. graminearum.

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