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A novel statin-mediated "prenylation block-and-release" assay provides insight into the membrane targeting mechanisms of small GTPases.

Ali BR, Nouvel I, Leung KF, Hume AN, Seabra MC - Biochem. Biophys. Res. Commun. (2010)

Bottom Line: To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus.We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins.However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates. bassam.ali@uaeu.ac.ae

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Investigation of the membrane targeting kinetics of GTPases that are substrates of different prenylation pathways following mevastatin wash-out. AtT20 cells were transiently transfected with plasmids allowing the expression of the indicated EGFP-fusion protein and incubated overnight with 5 μM mevastatin. The intracellular localization of the heterologously expressed protein was observed using confocal fluorescence microscopy at the indicated time-points following mevastatin wash-out. Bars = 10 μm.
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fig4: Investigation of the membrane targeting kinetics of GTPases that are substrates of different prenylation pathways following mevastatin wash-out. AtT20 cells were transiently transfected with plasmids allowing the expression of the indicated EGFP-fusion protein and incubated overnight with 5 μM mevastatin. The intracellular localization of the heterologously expressed protein was observed using confocal fluorescence microscopy at the indicated time-points following mevastatin wash-out. Bars = 10 μm.

Mentions: Finally we investigated whether the type (farnesyl versus geranylgeranyl) i.e. prenylation by different prenyl transferases, or the number of lipids (one or two) associated with a GTPase influences sensitivity to mevastatin and the mechanism of membrane targeting. As mentioned above most Rabs contain two prenylatable cysteine residues at their extreme C-terminus and are di-geranylgeranylated by RGGT [4]. However, a significant number including Rab23 have only one such cysteine. Also as described in the introduction other small GTPases e.g. Rho and Ras proteins, are singly prenylated by either GGT or FT. To address these issues we transfected mouse pituitary derived cell line (AtT20) with EGFP-H-Ras, EGFP-Rab23 (C-terminus tetra-peptide CSVP), predicted to be singly prenylated by FT and RGGT, respectively, and two Rab23 mutants; EGFP-Rab23-GGCC, that may be doubly prenylated by RGGT and EGFP-Rab23-CVLS, predicted to be a substrate for FT. We then tested the mevastatin sensitivity of prenylation and cytosol to membrane targeting of each of these proteins. As previously reported we found that all four proteins were targeted to the plasma membrane in the absence of mevastatin [25,28] (Fig. 3 untreated). Meanwhile addition of different concentrations of mevastatin revealed that targeting of Rab23 and Rab23-GGCC mutant (0.1 μM mevastatin disrupts targeting) were more sensitive to mevastatin than either Rab23-CVLS or H-Ras (5 μM and 1.6 μM mevastatin disrupt targeting, respectively). These data indicate that prenylation/targeting of FT substrates is less sensitive to mevastatin than RGGT substrates. We next investigated the recovery kinetics of prenylation/membrane targeting of GTPases that are substrates of different prenyltransferases. Consistent with the higher mevastatin sensitivity of RGGT compared with FT we found that recovery of cytosol to plasma membrane targeting was significantly slower for RGGT substrates, Rab23 wild-type and Rab23-GGCC, than for FT substrates Rab23-CVLS and H-Ras (Fig. 4, 90 min versus 30 min, respectively).


A novel statin-mediated "prenylation block-and-release" assay provides insight into the membrane targeting mechanisms of small GTPases.

Ali BR, Nouvel I, Leung KF, Hume AN, Seabra MC - Biochem. Biophys. Res. Commun. (2010)

Investigation of the membrane targeting kinetics of GTPases that are substrates of different prenylation pathways following mevastatin wash-out. AtT20 cells were transiently transfected with plasmids allowing the expression of the indicated EGFP-fusion protein and incubated overnight with 5 μM mevastatin. The intracellular localization of the heterologously expressed protein was observed using confocal fluorescence microscopy at the indicated time-points following mevastatin wash-out. Bars = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908739&req=5

fig4: Investigation of the membrane targeting kinetics of GTPases that are substrates of different prenylation pathways following mevastatin wash-out. AtT20 cells were transiently transfected with plasmids allowing the expression of the indicated EGFP-fusion protein and incubated overnight with 5 μM mevastatin. The intracellular localization of the heterologously expressed protein was observed using confocal fluorescence microscopy at the indicated time-points following mevastatin wash-out. Bars = 10 μm.
Mentions: Finally we investigated whether the type (farnesyl versus geranylgeranyl) i.e. prenylation by different prenyl transferases, or the number of lipids (one or two) associated with a GTPase influences sensitivity to mevastatin and the mechanism of membrane targeting. As mentioned above most Rabs contain two prenylatable cysteine residues at their extreme C-terminus and are di-geranylgeranylated by RGGT [4]. However, a significant number including Rab23 have only one such cysteine. Also as described in the introduction other small GTPases e.g. Rho and Ras proteins, are singly prenylated by either GGT or FT. To address these issues we transfected mouse pituitary derived cell line (AtT20) with EGFP-H-Ras, EGFP-Rab23 (C-terminus tetra-peptide CSVP), predicted to be singly prenylated by FT and RGGT, respectively, and two Rab23 mutants; EGFP-Rab23-GGCC, that may be doubly prenylated by RGGT and EGFP-Rab23-CVLS, predicted to be a substrate for FT. We then tested the mevastatin sensitivity of prenylation and cytosol to membrane targeting of each of these proteins. As previously reported we found that all four proteins were targeted to the plasma membrane in the absence of mevastatin [25,28] (Fig. 3 untreated). Meanwhile addition of different concentrations of mevastatin revealed that targeting of Rab23 and Rab23-GGCC mutant (0.1 μM mevastatin disrupts targeting) were more sensitive to mevastatin than either Rab23-CVLS or H-Ras (5 μM and 1.6 μM mevastatin disrupt targeting, respectively). These data indicate that prenylation/targeting of FT substrates is less sensitive to mevastatin than RGGT substrates. We next investigated the recovery kinetics of prenylation/membrane targeting of GTPases that are substrates of different prenyltransferases. Consistent with the higher mevastatin sensitivity of RGGT compared with FT we found that recovery of cytosol to plasma membrane targeting was significantly slower for RGGT substrates, Rab23 wild-type and Rab23-GGCC, than for FT substrates Rab23-CVLS and H-Ras (Fig. 4, 90 min versus 30 min, respectively).

Bottom Line: To allow association with membranes Ras-related GTPases are post-translationally modified by covalent attachment of prenyl groups to conserved cysteine residues at or near their C-terminus.We found that all Rabs tested were targeted directly to the membrane on which they reside at steady-state and not via an intermediate location as reported for Ras and Rho proteins.However, we observed that the kinetics of cytosol to membrane targeting differed for each Rab tested.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates. bassam.ali@uaeu.ac.ae

Show MeSH
Related in: MedlinePlus