Cloning, mapping and characterization of the human RAB27A gene.

Choroideremia (CHM) is an X-linked retinal degenerative disease that results from mutations in Rab Escort Protein-1 (REP1). REP1 acts in the prenylation of Rab GTPases, regulators of intracellular protein trafficking. Rab27a is unique among Rabs in that it is selectively unprenylated in CHM cells, suggesting that the degenerative process in CHM may result from unprenylation and consequent loss-of-function of Rab27a. As a first step towards the analysis of the Rab27a protein in patients, we report here the characterization of the human RAB27A gene. The putative protein encoded by this gene shares 96% identity with the previously cloned rat homologue. The RAB27A gene comprises five coding exons and two non-coding exons, of which one is alternatively used, and spans approximately 65 kb of DNA. There are three alternative poly-A addition sites in the long 3' UTR and also six potential single-nucleotide polymorphisms. The gene is located on chromosome 15q15-21.1, as determined by fluorescent in situ hybridization, and between markers D15S209 and AFM321ZD5 by radiation hybrid mapping.

Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex.

Rab proteins are geranylgeranylated on one or two C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase). The reaction is dependent on a Rab-binding protein, termed Rab escort protein (REP). Here, we studied the role of REP in the geranylgeranylation reaction. We first characterized the interaction between REP and ungeranylgeranylated Rab using analytical ultracentrifugation and a fluorescence-based assay. We measured an equilibrium dissociation constant of 0.2 microM for the formation of a 1:1 REP-Rab complex and showed that this interaction relies mostly on ionic bonds and does not involve the two C-terminal cysteine residues. Second, we show that REP is required for recognition of Rab by RabGGTase and therefore that the REP-Rab complex is the true substrate for RabGGTase. Third, we show that free REP inhibits the geranylgeranylation reaction, suggesting that the complex is recognized by RabGGTase primarily via a REP-binding site. Our data suggest a model whereby REP behaves kinetically as an essential activator of the reaction.

Deficient geranylgeranylation of Ram/Rab27 in choroideremia.

Choroideremia, an X-linked form of retinal degeneration, results from defects in the Rab escort protein-1 (REP-1) gene. REP-1 and REP-2 assist in the attachment of geranylgeranyl groups to Rab GTPases, a modification essential for their action as molecular switches regulating intracellular vesicular transport. If Rabs that depend preferentially on REP-1 for prenylation exist, they will accumulate unprenylated in choroideremia cells. Using recombinant Rab geranylgeranyl transferase and REPs to label unprenylated cytosolic proteins, we identified one unprenylated protein in choroideremia lymphoblasts that was prenylated in vitro more efficiently by REP-1 than by REP-2. This protein was purified and identified as Ram (renamed Rab27), a previously cloned Rab of unknown function. Immunohistochemistry of rat retina showed that Ram/Rab27 is expressed in the pigment epithelium and choriocapillaris, the two retinal cell layers that degenerate earliest in choroideremia. These results raise the possibility that the retinal degeneration in choroideremia results from the deficient geranylgeranylation of Ram/Rab27 or a closely related protein.

In vivo farnesylation of rat rhodopsin kinase.

We have shown by intravitreal injection of [3H]mevalonolactone that a 65 kDa protein in rat photoreceptors is posttranslationally modified by farnesylation. We further identified this 65 kDa prenylated protein as rhodopsin kinase based on its affinity for photolyzed rhodopsin and its ability to autophosphorylate in the presence of [gamma-32P]ATP. The farnesylation of rhodopsin kinase may be important for correctly targeting this enzyme to the photoreceptor outer segments, allowing it to phosphorylate photolyzed rhodopsin efficiently.

In vivo differential prenylation of retinal cyclic GMP phosphodiesterase catalytic subunits.

A number of phototransducing proteins in vertebrate photoreceptors contain a carboxyl terminal -CXXX motif (where C = cysteine and X = any amino acid), known to be a signal sequence for their post-translational prenylation and carboxyl methylation. To study the roles of these modifications in the visual excitation process, we have utilized an intravitreal injection method to radiolabel the prenylated proteins of rat retinas in vivo. We showed that two of the major prenylated polypeptides in the rod outer segments are the PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase (PDE). By chromatographic analyses of the amino acid constituents generated by exhaustive proteolysis of PDE alpha and PDE beta, we further demonstrated that they are differentially prenylated by farnesylation and geranylgeranylation, respectively. While a number of proteins ending with the -CXXX sequence have already been reported to possess either a farnesyl or a geranylgeranyl group, PDE is the first enzyme shown to be modified by both types of prenyl groups. The prenyl modification of PDE most likely plays a major role in membrane attachment and in correctly positioning the PDE molecule for phototransduction.