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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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Related in: MedlinePlus

Design of the prenylation block-and-release assay. (A–B) Cells were initially incubated with transfection complexes containing EGFP-GTPase encoding plasmid. Removal of complexes after 90 min and replacement with medium containing mevastatin (B–C ‘BLOCK’) allows uptake of the plasmid and initiation of EGFP-GTPase expression (indicated by pale green color of cytosol in B) but prevents association of EGFP-GTPase with intracellular membranes due to block of prenylation (indicated by black color of organelles in B). Overnight incubation with mevastatin allows cytosolic accumulation of EGFP-GTPase fusion protein (indicated by dark green color of cytosol in C). (C–D ‘RELEASE’) removal of mevastatin containing medium and incubation with normal growth medium allows resumption of prenylation and membrane targeting of the EGFP-GTPase (indicated by transition of cytosol from dark green (C) to pale green (D) and acquisition of green color by cytosolic organelles (D)). In some experiments (Fig. 2 2C) ‘BLOCK’ medium was supplemented by addition of GGPP precursor GGOH to confirm that the mevastatin specifically blocks membrane targeting of EGFP-GTPases by inhibition of prenylation. In other experiments (Fig. 2D) ‘RELEASE’ medium was supplemented with protein synthesis inhibitor cycloheximide to exclude the possibility that organelle associated protein was newly synthesized during the ‘RELEASE’ rather than protein accumulated during the mevastatin ‘BLOCK’.
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d32e563: Design of the prenylation block-and-release assay. (A–B) Cells were initially incubated with transfection complexes containing EGFP-GTPase encoding plasmid. Removal of complexes after 90 min and replacement with medium containing mevastatin (B–C ‘BLOCK’) allows uptake of the plasmid and initiation of EGFP-GTPase expression (indicated by pale green color of cytosol in B) but prevents association of EGFP-GTPase with intracellular membranes due to block of prenylation (indicated by black color of organelles in B). Overnight incubation with mevastatin allows cytosolic accumulation of EGFP-GTPase fusion protein (indicated by dark green color of cytosol in C). (C–D ‘RELEASE’) removal of mevastatin containing medium and incubation with normal growth medium allows resumption of prenylation and membrane targeting of the EGFP-GTPase (indicated by transition of cytosol from dark green (C) to pale green (D) and acquisition of green color by cytosolic organelles (D)). In some experiments (Fig. 2 2C) ‘BLOCK’ medium was supplemented by addition of GGPP precursor GGOH to confirm that the mevastatin specifically blocks membrane targeting of EGFP-GTPases by inhibition of prenylation. In other experiments (Fig. 2D) ‘RELEASE’ medium was supplemented with protein synthesis inhibitor cycloheximide to exclude the possibility that organelle associated protein was newly synthesized during the ‘RELEASE’ rather than protein accumulated during the mevastatin ‘BLOCK’.


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)

Design of the prenylation block-and-release assay. (A–B) Cells were initially incubated with transfection complexes containing EGFP-GTPase encoding plasmid. Removal of complexes after 90 min and replacement with medium containing mevastatin (B–C ‘BLOCK’) allows uptake of the plasmid and initiation of EGFP-GTPase expression (indicated by pale green color of cytosol in B) but prevents association of EGFP-GTPase with intracellular membranes due to block of prenylation (indicated by black color of organelles in B). Overnight incubation with mevastatin allows cytosolic accumulation of EGFP-GTPase fusion protein (indicated by dark green color of cytosol in C). (C–D ‘RELEASE’) removal of mevastatin containing medium and incubation with normal growth medium allows resumption of prenylation and membrane targeting of the EGFP-GTPase (indicated by transition of cytosol from dark green (C) to pale green (D) and acquisition of green color by cytosolic organelles (D)). In some experiments (Fig. 2 2C) ‘BLOCK’ medium was supplemented by addition of GGPP precursor GGOH to confirm that the mevastatin specifically blocks membrane targeting of EGFP-GTPases by inhibition of prenylation. In other experiments (Fig. 2D) ‘RELEASE’ medium was supplemented with protein synthesis inhibitor cycloheximide to exclude the possibility that organelle associated protein was newly synthesized during the ‘RELEASE’ rather than protein accumulated during the mevastatin ‘BLOCK’.
© Copyright Policy
Related In: Results  -  Collection

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

d32e563: Design of the prenylation block-and-release assay. (A–B) Cells were initially incubated with transfection complexes containing EGFP-GTPase encoding plasmid. Removal of complexes after 90 min and replacement with medium containing mevastatin (B–C ‘BLOCK’) allows uptake of the plasmid and initiation of EGFP-GTPase expression (indicated by pale green color of cytosol in B) but prevents association of EGFP-GTPase with intracellular membranes due to block of prenylation (indicated by black color of organelles in B). Overnight incubation with mevastatin allows cytosolic accumulation of EGFP-GTPase fusion protein (indicated by dark green color of cytosol in C). (C–D ‘RELEASE’) removal of mevastatin containing medium and incubation with normal growth medium allows resumption of prenylation and membrane targeting of the EGFP-GTPase (indicated by transition of cytosol from dark green (C) to pale green (D) and acquisition of green color by cytosolic organelles (D)). In some experiments (Fig. 2 2C) ‘BLOCK’ medium was supplemented by addition of GGPP precursor GGOH to confirm that the mevastatin specifically blocks membrane targeting of EGFP-GTPases by inhibition of prenylation. In other experiments (Fig. 2D) ‘RELEASE’ medium was supplemented with protein synthesis inhibitor cycloheximide to exclude the possibility that organelle associated protein was newly synthesized during the ‘RELEASE’ rather than protein accumulated during the mevastatin ‘BLOCK’.
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