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. email@example.comShow MeSH
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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 . 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).
Affiliation: Department of Pathology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates. firstname.lastname@example.org