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Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

Wen L, Fukuda M, Sunada M, Ishino S, Ishino Y, Okita TW, Ogawa M, Ueda T, Kumamaru T - J. Exp. Bot. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan Tobacco Research Institute, Chinese Academy of Agricultural Science, Qingdao 266101, China.

No MeSH data available.


Related in: MedlinePlus

in vitro GEF assay of truncated GEFs. The conformational changes of Rab5s and Rab11 (as 1 μM GST fusion proteins) upon GDP/GMP-PNP exchange were measured by monitoring Trp autofluorescence when incubated with 0 μM (blue), 0.25 μM (magenta), 0.5μM (yellow), and 1 μM (red) GST-truncate GEFs. (A) Schematic structure of GEF proteins. VPS9a: Rab5 specific GEF in Arabidopsis (Goh et al., 2007). Δhelical bundle of Rab5-GEF2: deletion of helical bundle domain sequence, 90 amino acids from Rab5-GEF2. Δc-terminal of GLUP6/GEF: deletion of C-terminal sequence, 162 amino acids from GLUP6/GEF. (B) in vitro GEF assay of Δc-terminal of GLUP6/GEF. Note that GST-Δc-terminal of GLUP6/GEF activates GLUP4/Rab5a and Rab5c. (C) in vitro GEF assay of Δhelical bundle, Δc-terminal of GLUP6/GEF. Note that GST-Δhelical bundle, Δc-terminal of GLUP6/GEF cannot activate GLUP4/Rab5a, Rab5b and Rab5c. (D) in vitro GEF assay of Δhelical bundle of Rab5-GEF2. Note that GST-Δhelical bundle of Rab5-GEF2 cannot activate GLUP4/Rab5a, Rab5b, and Rab5c.
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Figure 8: in vitro GEF assay of truncated GEFs. The conformational changes of Rab5s and Rab11 (as 1 μM GST fusion proteins) upon GDP/GMP-PNP exchange were measured by monitoring Trp autofluorescence when incubated with 0 μM (blue), 0.25 μM (magenta), 0.5μM (yellow), and 1 μM (red) GST-truncate GEFs. (A) Schematic structure of GEF proteins. VPS9a: Rab5 specific GEF in Arabidopsis (Goh et al., 2007). Δhelical bundle of Rab5-GEF2: deletion of helical bundle domain sequence, 90 amino acids from Rab5-GEF2. Δc-terminal of GLUP6/GEF: deletion of C-terminal sequence, 162 amino acids from GLUP6/GEF. (B) in vitro GEF assay of Δc-terminal of GLUP6/GEF. Note that GST-Δc-terminal of GLUP6/GEF activates GLUP4/Rab5a and Rab5c. (C) in vitro GEF assay of Δhelical bundle, Δc-terminal of GLUP6/GEF. Note that GST-Δhelical bundle, Δc-terminal of GLUP6/GEF cannot activate GLUP4/Rab5a, Rab5b and Rab5c. (D) in vitro GEF assay of Δhelical bundle of Rab5-GEF2. Note that GST-Δhelical bundle of Rab5-GEF2 cannot activate GLUP4/Rab5a, Rab5b, and Rab5c.

Mentions: The deduced primary sequence of Rab5-GEF2, 308 amino acids in length, is 172 residues shorter than GLUP6/GEF as it lacks the longer C-terminal region of GLUP6/GEF (Fig. 8A). As Rab5-GEF2 activates Rab proteins, the C-terminal region in GLUP6/GEF is likely not required for activation of the Rab5 proteins. To obtain evidence to support this view, a truncated form of GLUP6/GEF (ΔC-GLUP6/GEF) was constructed, which lacks 162 amino acids at the C-terminal region (Fig. 8A). As shown in Fig. 8B, ΔC-GLUP6/GEF was capable of activating GLUP4/Rab5a and Rab5c in a dose-dependent manner, indicating that the C-terminal region is not required for activation of Rab5 proteins.


Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

Wen L, Fukuda M, Sunada M, Ishino S, Ishino Y, Okita TW, Ogawa M, Ueda T, Kumamaru T - J. Exp. Bot. (2015)

in vitro GEF assay of truncated GEFs. The conformational changes of Rab5s and Rab11 (as 1 μM GST fusion proteins) upon GDP/GMP-PNP exchange were measured by monitoring Trp autofluorescence when incubated with 0 μM (blue), 0.25 μM (magenta), 0.5μM (yellow), and 1 μM (red) GST-truncate GEFs. (A) Schematic structure of GEF proteins. VPS9a: Rab5 specific GEF in Arabidopsis (Goh et al., 2007). Δhelical bundle of Rab5-GEF2: deletion of helical bundle domain sequence, 90 amino acids from Rab5-GEF2. Δc-terminal of GLUP6/GEF: deletion of C-terminal sequence, 162 amino acids from GLUP6/GEF. (B) in vitro GEF assay of Δc-terminal of GLUP6/GEF. Note that GST-Δc-terminal of GLUP6/GEF activates GLUP4/Rab5a and Rab5c. (C) in vitro GEF assay of Δhelical bundle, Δc-terminal of GLUP6/GEF. Note that GST-Δhelical bundle, Δc-terminal of GLUP6/GEF cannot activate GLUP4/Rab5a, Rab5b and Rab5c. (D) in vitro GEF assay of Δhelical bundle of Rab5-GEF2. Note that GST-Δhelical bundle of Rab5-GEF2 cannot activate GLUP4/Rab5a, Rab5b, and Rab5c.
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Figure 8: in vitro GEF assay of truncated GEFs. The conformational changes of Rab5s and Rab11 (as 1 μM GST fusion proteins) upon GDP/GMP-PNP exchange were measured by monitoring Trp autofluorescence when incubated with 0 μM (blue), 0.25 μM (magenta), 0.5μM (yellow), and 1 μM (red) GST-truncate GEFs. (A) Schematic structure of GEF proteins. VPS9a: Rab5 specific GEF in Arabidopsis (Goh et al., 2007). Δhelical bundle of Rab5-GEF2: deletion of helical bundle domain sequence, 90 amino acids from Rab5-GEF2. Δc-terminal of GLUP6/GEF: deletion of C-terminal sequence, 162 amino acids from GLUP6/GEF. (B) in vitro GEF assay of Δc-terminal of GLUP6/GEF. Note that GST-Δc-terminal of GLUP6/GEF activates GLUP4/Rab5a and Rab5c. (C) in vitro GEF assay of Δhelical bundle, Δc-terminal of GLUP6/GEF. Note that GST-Δhelical bundle, Δc-terminal of GLUP6/GEF cannot activate GLUP4/Rab5a, Rab5b and Rab5c. (D) in vitro GEF assay of Δhelical bundle of Rab5-GEF2. Note that GST-Δhelical bundle of Rab5-GEF2 cannot activate GLUP4/Rab5a, Rab5b, and Rab5c.
Mentions: The deduced primary sequence of Rab5-GEF2, 308 amino acids in length, is 172 residues shorter than GLUP6/GEF as it lacks the longer C-terminal region of GLUP6/GEF (Fig. 8A). As Rab5-GEF2 activates Rab proteins, the C-terminal region in GLUP6/GEF is likely not required for activation of the Rab5 proteins. To obtain evidence to support this view, a truncated form of GLUP6/GEF (ΔC-GLUP6/GEF) was constructed, which lacks 162 amino acids at the C-terminal region (Fig. 8A). As shown in Fig. 8B, ΔC-GLUP6/GEF was capable of activating GLUP4/Rab5a and Rab5c in a dose-dependent manner, indicating that the C-terminal region is not required for activation of Rab5 proteins.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan Tobacco Research Institute, Chinese Academy of Agricultural Science, Qingdao 266101, China.

No MeSH data available.


Related in: MedlinePlus