A novel statin-mediated "prenylation block-and-release" assay provides insight into the membrane targeting mechanisms of small GTPases.
Bottom Line: 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.Comparison of the mevastatin sensitivity and kinetics of membrane targeting of Rab23, Rab23 prenylation motif mutants and H-Ras revealed that these parameters are strongly dependent upon the prenyl transferase with Rab geranylgeranyl transferase substrates exhibiting higher sensitivity and requiring greater time to recover from mevastatin inhibition than farnesyl transferase substrates.
Affiliation: Department of Pathology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates. firstname.lastname@example.orgShow MeSH
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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) . 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 .
Affiliation: Department of Pathology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates. email@example.com