Abstract
Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs. However, very little is known about processing and membrane targeting of Rabs that naturally contain a CAAX motif. We show here that a variety of Rab-CAAX proteins undergo carboxyl methylation, both in vitro and in vivo, with one exception. Rab38(CAKS) is not methylated in vivo, presumably because of the inhibitory action of the lysine residue within the AAX motif for cleavage by Rce1. Unlike farnesylated Ras proteins, we observed no targeting defects of overexpressed Rab-CAAX proteins in cells deficient in Rce1 or Icmt, as reported for geranylgeranylated Rho proteins. However, endogenous geranylgeranylated non-methylated Rab-CAAX and Rab-CXC proteins were significantly redistributed to the cytosol at steady-state levels and redistribution correlates with higher affinity of RabGDI for non-methylated Rabs in Icmt-deficient cells. Our data suggest a role for methylation in Rab function by regulating the cycle of Rab membrane recruitment and retrieval. Our findings also imply that those Rabs that undergo post-prenylation processing follow an indirect targeting pathway requiring initial endoplasmic reticulum membrane association prior to specific organelle targeting.
Highlights
Out intracellular functions [1, 2]
The newly exposed prenylated cysteine is further modified by carboxyl methylation on the ␣-carboxyl group by isoprenylcysteine carboxyl methyltransferase (Icmt), which is located on the endoplasmic reticulum (ER) [6, 7]
In Vitro Carboxyl Methylation of Rab-CAAX Proteins—To determine whether Rab proteins that naturally possess a CAAX motif are substrates for Rce1 and Icmt in vitro, recombinant GST-Rab fusion proteins were produced after expression in Escherichia coli
Summary
Plasmid Constructs—pEGFP-Rab11a was a kind gift of James Goldenring (Vanderbilt University School of Medicine, Nashville, TN). pEGFP-mRab was a kind gift from Carol Wicking (University of Queensland, Australia) (from here on, mRab will be referred to as Rab23). pEGFP-Rab8aGGCC, pEGFPRab8aGCSC, pEGFP-Rab38CALS, and pEGFP-Rab38CAVS were generated using the Stratagene QuikChange site-directed mutagenesis system, as described previously [15]. pGEX-4T-1Rab, Rab, and Rab were generated by PCR amplification of the Rab cDNA of interest and cloned into pGEX-4T-1 vector using EcoRI-SalI, EcoRI-XhoI, and EcoRI-XhoI, respectively. The cells were resuspended in 200 l of RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5% sodium deoxycholate) containing complete protease inhibitor mixture (Roche) for 10 min to allow lysis to occur This was followed by centrifugation at 10,000 ϫ g and the post-nuclear supernatant (PNS) was transferred to a fresh tube. RabGDI Extraction Assay—Membrane proteins (30 g) prepared from MEFs in buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM dithioerythritol, 1 mM GDP, and Roche complete protease inhibitor mixture, were incubated with increasing amounts of purified His-RabGDI (0 – 8 M) for 20 min at 37 °C. The samples (50 l) were injected, and the material eluting between 0.8 and 2 ml was collected in 50-l fractions
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