RESUMO
Rice glutelin polypeptides are initially synthesized on the endoplasmic reticulum (ER) membrane as a proglutelin, which are then transported to the protein storage vacuole (PSV) via the Golgi apparatus. Rab5 and its cognate activator guanine nucleotide exchange factor (GEF) are essential for the intracellular transport of proglutelin from the Golgi apparatus to the PSV. Results from previous studies showed that the double recessive type of glup4/rab5a and glup6/gef mutant accumulated much higher amounts of proglutelin than either parent line. The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect. These observations led us to the hypothesis that other isoforms of Rab5 and GEF also participate in the intracellular transport of rice glutelin. A database search identified a novel guanine nucleotide exchange factor, Rab5-GEF2. Like GLUP6/GEF, Rab5-GEF2 was capable of activating Rab5a and two other Rab5 isoforms in in vitro GTP/GDP exchange assays. GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region. By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins.
Assuntos
Glutens/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Endosperma , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Dados de Sequência Molecular , Mutação , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Transporte Proteico/genética , Análise de Sequência de DNA , Vacúolos/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
We describe a high-throughput screening (HTS)-compatible method for detecting GTPase exchange factor (GEF) activity based on stimulation of GDP formation by Rho GTPases. The method is based on the fact that GDP dissociation is the rate-limiting step in the Rho GTPase catalytic cycle, so by accelerating its release a GEF causes an increase in the steady-state rate of GDP formation. The Transcreener® GDP GTPase Assay, a fluorescence polarization immunoassay (FPIA), is used to detect GDP formation in a homogeneous format.
Assuntos
Guanosina Difosfato/química , Fatores de Troca de Nucleotídeo Guanina Rho/química , Proteínas rho de Ligação ao GTP/química , Imunoensaio de Fluorescência por Polarização/métodos , Guanosina Difosfato/metabolismo , Humanos , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
GTPases are molecular switches that cycle between the inactive GDP-bound state and the active GTP-bound state. GTPases exchange nucleotides either by its intrinsic nucleotide exchange or by interaction with guanine nucleotide exchange factors (GEFs). Monitoring the nucleotide exchange in vitro, together with reconstitution of direct interactions with regulatory proteins, provides key insights into how a GTPase is activated. In this protocol, we describe core methods to monitor nucleotide exchange using fluorescent N-Methylanthraniloyl (MANT)-guanine nucleotide.