Farnesylation carboxylmethylation

A. The Study of Obscure Fungal Pheromones Led to the Discovery OF Farnesylation and Carboxylmethylation as POSTTRANSLATIONAL MODIFICATIONS OF THE C-TeRMINAL CySTEINE... 

Mature a-factor is a tiny (1.6 kDa) farnesylated and carboxylmethylated 12-mer peptide. Both its small size and hydrophobic nature, due in large part to its CAAX modifications, present some unique challenges for biochemical manipulation and qnantitation. One challenge in working with either intracellular a-factor intermediates or extracellular mature a-factor is its stickiness that canses adherence to plastic tubes and pipette tips. This can be avoided by inclnding excess BSA to coat surfaces and keep a-factor suspended [18,46]. 

Fig. 4.3. Farnesylated CaaX proteins are more dependent on carboxylmethylation by Icmt for correct membrane localization than are geranylgeranylated CaaX proteins. (A) The indicated GFP-tagged Ras isoform was expressed in MEFs deficient in Icmt (-/-) or wild-type MEFs from httermates (+/+). All three Ras isoforms showed marked mislocalization with accumulation of GFP-Ras in the fluid phase cytosol. (B) GFP-tagged Rho proteins were expressed in the same MEFs as in (A). Activated forms of Racl, RhoA, and Cdc42 were utilized for clarity since these forms do not bind to the cytosolic chaperone RhoGDI but instead associate with the cellular membranes to which their C-termini are targeted. In contrast to farnesylated Ras proteins, geranylgeranylated Rho proteins are localized with similar patterns in both +/+ and -/- MEFs.

Fig. 10.1. Schematic of CaaX protein biosynthesis. A precursor containing a CaaX motif is sequentially modified to yield a product with an isoprenylated and carboxylmethylated C-ter-minus. Rcelp is specific for Ras substrates and partially redundant with Ste24p in mediating proteolysis of certain other CaaX proteins (step 2). FTI, farnesyl transferase inhibitor RPI, Rcelp protease inhibitor.

GGTase-I [5], The specificities of FTase and GGTase are not absolute, and cross prenylation occurs which can be observed more readily for some substrates when one of the enzyme is inhibited. For example, K-Ras and N-Ras, which are the usual substrates for FTase, can be geranylgeranylated when the activity of FTase is suppressed [6]. The second and last steps of the modification process occur on the ER membrane and are catalyzed by Ras converting enzyme 1 (Reel) and isoprenylcysteine carboxylmethyl transferase (Icmt), both unique enzymes modify both farnesylated and geranylgeranylated proteins [1,7]. 

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