Functional Overlap between Retinitis Pigmentosa 2 Protein and the Tubulin-specific Chaperone Cofactor C

Abstract

Mutations in the X-linked retinitis pigmentosa 2 gene cause progressive degeneration of photoreceptor cells. The retinitis pigmentosa 2 protein (RP2) is similar in sequence to the tubulin-specific chaperone cofactor C. Together with cofactors D and E, cofactor C stimulates the GTPase activity of native tubulin, a reaction regulated by ADP-ribosylation factor-like 2 protein. Here we show that in the presence of cofactor D, RP2 protein also stimulates the GTPase activity of tubulin. We find that this function is abolished by mutation in an arginine residue that is conserved in both cofactor C and RP2. Notably, mutations that alter this arginine codon cause familial retinitis pigmentosa. Our data imply that this residue acts as an "arginine finger" to trigger the tubulin GTPase activity and suggest that loss of this function in RP2 contributes to retinal degeneration. We also show that in Saccharomyces cerevisiae, both cofactor C and RP2 partially complement the microtubule phenotype resulting from deletion of the cofactor C homolog, demonstrating their functional overlap in vivo. Finally, we find that RP2 interacts with GTP-bound ADP ribosylation factor-like 3 protein, providing a link between RP2 and several retinal-specific proteins, mutations in which also cause retinitis pigmentosa.Retinitis pigmentosa is a degenerative disease and the major cause of heritable blindness (1). It is caused by mutations in a dizzying variety of genes; some are structural proteins of photoreceptors, many are involved in photoreception and transduction, and a few are seemingly unrelated ubiquitously expressed proteins (see RetNet, www.sph.uth.tmc.edu/retnet/). Most (80 -90%) cases of X-linked retinitis pigmentosa are caused by mutations in two genes, XRP2 and XRP3 (2). These were isolated by positional cloning and shown to encode, respectively, the RP2 1 protein (3) and the retinitis pigmentosa GTPase regulator (RP3) (4, 5). The function of RP2 is unknown, and the gene is expressed at a low level in all tissues examined; it is targeted to the plasma membrane by myristoylation and palmitoylation of its amino terminus (6).RP2 has amino acid sequence similarity over half of its length to the tubulin-specific chaperone protein cofactor C (3). It has been shown that cofactor C acts in a pathway together with four other tubulin-specific chaperone proteins (cofactors A, B, D, and E) to chaperone quasi-native ␣-and ␤-tubulin subunits released from the cytosolic chaperonin CCT and assemble the ␣/␤-tubulin heterodimer. Each step in this pathway has been deduced from in vitro reconstitution experiments using purified components (7-9). The pathway hinges on the formation of a supercomplex containing ␣Ϫ and ␤-tubulin and cofactors C, D, and E. The hydrolysis of GTP by tubulin, stimulated by cofactors C and D, is part of the heterodimer assembly reaction; the stimulated hydrolysis of GTP by ␤-tubulin acts as a switch for the release from the supercomplex of native, newly made tubulin heterodimers (10). Cofactor C and D in combination have also b...

Genetic disorders involving molecular-chaperone genes: A perspective

“…47 These and other data from experiments with Saccharomyces cerevisiae indicated that RP2 and cofactor C have overlapping functions, in addition to other functions that are specific to each molecule. It was also shown that RP2 reacts with ADPribosylation factor-like 3-protein, when the latter protein is bound to GTP.…”

“…46 The N-terminal segment of RP2 has similarity with cofactor C, which in humans is one of a set of at least five chaperones (A through E) involved in tubulin folding. 46,47 Cofactors C and D activate tubulin GTPase (hence they are called GTPase activator proteins, or GAP). RP2, together with cofactor D, serves as the GAP for tubulin, meaning that RP2 can replace cofactor C. Although RP2 replaced cofactor C to act as GAP in conjunction with cofactor D, RP2 did not catalyze tubulin heterodimerization as cofactor C did.…”

Revisiting the tubulin folding pathway: new roles in centrosomes and cilia

“…In a yeast complementation assay, RP2 was shown to partially complement the CIN2 (tbcc homolog) deletion. However, unlike TBCC, RP2 does not promote tubulin heterodimerization in vitro (111). The GAP activity of RP2 and TBCC relies on the TBCC domain and many of the RP2 mutations involved in the retinitis pigmentosa pathology occur in this domain (109).…”

Photoreceptor Sensory Cilium and Associated Disorders

“…The structure of RP2 reveals two major domains: an amino-terminal domain homologous to tubulin binding cofactor C (TBCC) homology domain and a carboxyl-terminal nucleoside diphosphate kinase domain [77][78][79] (Figure 3). The purified RP2 protein possesses GTPase activating protein (GAP) activity towards the small GTPase ARL3 (ADP Ribosylation FactorLike protein 3).…”