A novel statin-mediated "prenylation block-and-release" assay provides insight into the membrane targeting mechanisms of small GTPases.
Ras super-family small GTPases regulate diverse cellular processes such as vesicular transport and signal transduction. Critical to these activities is the ability of these proteins to target to specific intracellular membranes. 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. Here we used the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase (HMGCR) inhibitor mevastatin to develop a 'prenylation block-and-release' assay that allows membrane targeting of prenylated proteins to be visualized in living cells. Using this assay we investigated the cytosol to membrane targeting of several small GTPases to compartments of the secretory and endocytic pathways. 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. We propose that this assay is a useful tool to investigate the kinetics, biological functions and the mechanisms of membrane targeting of prenylated proteins.
Ali
Bassam R
BR
Department of Pathology, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates. bassam.ali@uaeu.ac.ae
Nouvel
Ian
I
Leung
Ka Fai
KF
Hume
Alistair N
AN
Seabra
Miguel C
MC
D002462:Cell Membrane / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D008148:Lovastatin / Q000031:analogs & derivatives* / Q000494:pharmacology; D017368:Protein Prenylation / Q000187:drug effects*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C012258:mevastatin; D008148:Lovastatin; C486614:RAB23 protein, human; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D018631:ras Proteins
Curr Opin Genet Dev. 2005 Feb;15(1):62-8
15661535
EMBO J. 1994 Nov 15;13(22):5262-73
7957092
N Engl J Med. 2005 May 26;352(21):2184-92
15917383
J Cell Biol. 2001 Feb 19;152(4):795-808
11266470
J Biol Chem. 1996 Mar 8;271(10):5289-92
8621375
Biochim Biophys Acta. 2006 Dec;1764(12):1914-31
17110180
Front Biosci. 2007 May 01;12:4456-72
17485388
Mol Biol Cell. 2003 May;14(5):1882-99
12802062
Science. 2001 May 11;292(5519):1160-4
11349148
J Cell Biol. 1992 Nov;119(4):749-61
1429835
J Cell Biol. 2001 Jan 8;152(1):111-26
11149925
Cell. 1991 Mar 8;64(5):915-25
1900457
J Lipid Res. 2006 Mar;47(3):467-75
16401880
J Mol Biol. 2000 Aug 25;301(4):1077-87
10966806
J Cell Sci. 2004 Dec 15;117(Pt 26):6401-12
15561774
FEBS Lett. 2001 Jun 8;498(2-3):197-200
11412856
Traffic. 2003 Dec;4(12):869-84
14617350
Cell Signal. 1998 Mar;10(3):167-72
9607139
J Lipid Res. 2006 May;47(5):883-91
16543601
J Biol Chem. 2003 Nov 21;278(47):46798-804
12941939
Cell. 1999 Jul 9;98(1):69-80
10412982
Prog Med Chem. 2002;40:1-22
12516521
Pharmacol Res. 1995 Jan;31(1):9-27
7784310
Cell. 1989 Jun 30;57(7):1167-77
2661017
Nat Rev Mol Cell Biol. 2009 Aug;10(8):513-25
19603039
Biochem J. 1998 Aug 1;333 ( Pt 3):497-504
9677305
J Lipid Res. 1992 Nov;33(11):1569-82
1464741
Biochem Med Metab Biol. 1992 Oct;48(2):149-58
1419147
J Biol Chem. 2007 Jan 12;282(2):1487-97
17114793