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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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Use of the mevastatin ‘prenylation-block-and-release assay’ to investigate the membrane targeting of EGFP-Rab1a and EGFP-Rab27a. HeLa cells and melan-ash melanocytes were transfected with pEGFP-Rab1a or pEGFP-Rab27a, respectively, treated with mevastatin overnight before the localization of EGFP-fusion protein was monitored during recovery from drug treatment. (A) shows EGFP-Rab1a (left) is targeted to Golgin-97 (middle) positive Golgi membranes in untreated cells. Co-localization of EGFP-Rab1a (green) and Golgin-97 (red) is indicated by yellow in the right-hand panel. (B) shows the localization of EGFP-Rab1a in transfected cells at indicated time-points following mevastatin (10 μM) wash-out. (C) shows the localization of EGFP-Rab27a and melanosomes (upper and lower panels, respectively) in melan-ash melanocytes in the absence, presence or 180 min after wash-out (left, middle and right, respectively) of mevastatin (1 μM). Arrows in phase contrast images indicate transfected cells. For right-hand panels the boxed area is enlarged and shown in the bottom right corner with arrows to indicate co-localization of EGFP-Rab27a with pigmented melanosome. (D) shows the time-course of recovery of membrane targeting of EGFP-H-Ras in HeLa cells following wash-out of mevastatin (10 μM) (white asterisks indicate the position of the Golgi and white arrows indicate plasma membrane associated H-Ras). Bars = 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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fig2: Use of the mevastatin ‘prenylation-block-and-release assay’ to investigate the membrane targeting of EGFP-Rab1a and EGFP-Rab27a. HeLa cells and melan-ash melanocytes were transfected with pEGFP-Rab1a or pEGFP-Rab27a, respectively, treated with mevastatin overnight before the localization of EGFP-fusion protein was monitored during recovery from drug treatment. (A) shows EGFP-Rab1a (left) is targeted to Golgin-97 (middle) positive Golgi membranes in untreated cells. Co-localization of EGFP-Rab1a (green) and Golgin-97 (red) is indicated by yellow in the right-hand panel. (B) shows the localization of EGFP-Rab1a in transfected cells at indicated time-points following mevastatin (10 μM) wash-out. (C) shows the localization of EGFP-Rab27a and melanosomes (upper and lower panels, respectively) in melan-ash melanocytes in the absence, presence or 180 min after wash-out (left, middle and right, respectively) of mevastatin (1 μM). Arrows in phase contrast images indicate transfected cells. For right-hand panels the boxed area is enlarged and shown in the bottom right corner with arrows to indicate co-localization of EGFP-Rab27a with pigmented melanosome. (D) shows the time-course of recovery of membrane targeting of EGFP-H-Ras in HeLa cells following wash-out of mevastatin (10 μM) (white asterisks indicate the position of the Golgi and white arrows indicate plasma membrane associated H-Ras). Bars = 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Mentions: We next used the prenylation block-and-release assay to investigate the targeting of other Rabs to organelles of the secretory pathway. To do this we examined the membrane targeting of EGFP-Rab1a and EGFP-Rab27a that regulate secretory pathway ER to Golgi transport and melanosome docking in peripheral melanocyte dendrites, respectively [13,27]. As for EGFP-Rab5a, we observed that in the untreated cells EGFP-Rab1a and EGFP-Rab27a were correctly targeted to the Golgi stack and mature pigmented melanosomes in melanocytes, respectively (Fig. 2A and C). Moreover, we confirm the functionality of EGFP-Rab27a as its association with melanosomes restores their peripheral retention in Rab27a (melan-ash) melanocytes (Fig. 2C) [11]. Mevastatin treatment of transfected cells resulted in a diffuse distribution of both EGFP-Rab1a and EGFP-Rab27a throughout the nucleus and cytoplasm consistent with the disruption of prenylation and membrane targeting (Fig. 2B and C). Also in the case of EGFP-Rab27a in melan-ash function is disrupted as evidence by maintenance of perinuclear clustered melanosomes in transfected cells (Fig. 2C). Subsequent removal of mevastatin resulted in membrane targeting of the newly prenylated protein. For EGFP-Rab1a protein was targeted from the cytosol to a ribbon-like perinuclear structure, consistent with the normal distribution of the Golgi stack, (Fig. 2B and Supplementary Fig. 2B and Movie 2) while EGFP-Rab27a was targeted from the cytosol to pigmented melanosomes and restored their retention in peripheral dendrites consistent with full functionality of the targeted protein (Fig. 2C). Interestingly while both Rabs targeted directly to these organelles we observed differences in the kinetics of recovery from the block for each Rab with EGFP-Rab1a and EGFP-Rab27a requiring 30 and 180 min, respectively. In contrast, following release of the mevastatin block EGFP-H-Ras was first observed on the Golgi complex within 30 min then accumulated at the plasma membrane at later time-points (Fig. 2D). This is consistent with previous reports showing that H-Ras is targeted to the plasma membrane via the classical secretory pathway [25].


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)

Use of the mevastatin ‘prenylation-block-and-release assay’ to investigate the membrane targeting of EGFP-Rab1a and EGFP-Rab27a. HeLa cells and melan-ash melanocytes were transfected with pEGFP-Rab1a or pEGFP-Rab27a, respectively, treated with mevastatin overnight before the localization of EGFP-fusion protein was monitored during recovery from drug treatment. (A) shows EGFP-Rab1a (left) is targeted to Golgin-97 (middle) positive Golgi membranes in untreated cells. Co-localization of EGFP-Rab1a (green) and Golgin-97 (red) is indicated by yellow in the right-hand panel. (B) shows the localization of EGFP-Rab1a in transfected cells at indicated time-points following mevastatin (10 μM) wash-out. (C) shows the localization of EGFP-Rab27a and melanosomes (upper and lower panels, respectively) in melan-ash melanocytes in the absence, presence or 180 min after wash-out (left, middle and right, respectively) of mevastatin (1 μM). Arrows in phase contrast images indicate transfected cells. For right-hand panels the boxed area is enlarged and shown in the bottom right corner with arrows to indicate co-localization of EGFP-Rab27a with pigmented melanosome. (D) shows the time-course of recovery of membrane targeting of EGFP-H-Ras in HeLa cells following wash-out of mevastatin (10 μM) (white asterisks indicate the position of the Golgi and white arrows indicate plasma membrane associated H-Ras). Bars = 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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fig2: Use of the mevastatin ‘prenylation-block-and-release assay’ to investigate the membrane targeting of EGFP-Rab1a and EGFP-Rab27a. HeLa cells and melan-ash melanocytes were transfected with pEGFP-Rab1a or pEGFP-Rab27a, respectively, treated with mevastatin overnight before the localization of EGFP-fusion protein was monitored during recovery from drug treatment. (A) shows EGFP-Rab1a (left) is targeted to Golgin-97 (middle) positive Golgi membranes in untreated cells. Co-localization of EGFP-Rab1a (green) and Golgin-97 (red) is indicated by yellow in the right-hand panel. (B) shows the localization of EGFP-Rab1a in transfected cells at indicated time-points following mevastatin (10 μM) wash-out. (C) shows the localization of EGFP-Rab27a and melanosomes (upper and lower panels, respectively) in melan-ash melanocytes in the absence, presence or 180 min after wash-out (left, middle and right, respectively) of mevastatin (1 μM). Arrows in phase contrast images indicate transfected cells. For right-hand panels the boxed area is enlarged and shown in the bottom right corner with arrows to indicate co-localization of EGFP-Rab27a with pigmented melanosome. (D) shows the time-course of recovery of membrane targeting of EGFP-H-Ras in HeLa cells following wash-out of mevastatin (10 μM) (white asterisks indicate the position of the Golgi and white arrows indicate plasma membrane associated H-Ras). Bars = 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Mentions: We next used the prenylation block-and-release assay to investigate the targeting of other Rabs to organelles of the secretory pathway. To do this we examined the membrane targeting of EGFP-Rab1a and EGFP-Rab27a that regulate secretory pathway ER to Golgi transport and melanosome docking in peripheral melanocyte dendrites, respectively [13,27]. As for EGFP-Rab5a, we observed that in the untreated cells EGFP-Rab1a and EGFP-Rab27a were correctly targeted to the Golgi stack and mature pigmented melanosomes in melanocytes, respectively (Fig. 2A and C). Moreover, we confirm the functionality of EGFP-Rab27a as its association with melanosomes restores their peripheral retention in Rab27a (melan-ash) melanocytes (Fig. 2C) [11]. Mevastatin treatment of transfected cells resulted in a diffuse distribution of both EGFP-Rab1a and EGFP-Rab27a throughout the nucleus and cytoplasm consistent with the disruption of prenylation and membrane targeting (Fig. 2B and C). Also in the case of EGFP-Rab27a in melan-ash function is disrupted as evidence by maintenance of perinuclear clustered melanosomes in transfected cells (Fig. 2C). Subsequent removal of mevastatin resulted in membrane targeting of the newly prenylated protein. For EGFP-Rab1a protein was targeted from the cytosol to a ribbon-like perinuclear structure, consistent with the normal distribution of the Golgi stack, (Fig. 2B and Supplementary Fig. 2B and Movie 2) while EGFP-Rab27a was targeted from the cytosol to pigmented melanosomes and restored their retention in peripheral dendrites consistent with full functionality of the targeted protein (Fig. 2C). Interestingly while both Rabs targeted directly to these organelles we observed differences in the kinetics of recovery from the block for each Rab with EGFP-Rab1a and EGFP-Rab27a requiring 30 and 180 min, respectively. In contrast, following release of the mevastatin block EGFP-H-Ras was first observed on the Golgi complex within 30 min then accumulated at the plasma membrane at later time-points (Fig. 2D). This is consistent with previous reports showing that H-Ras is targeted to the plasma membrane via the classical secretory pathway [25].

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