PMC

Subcellular compartmentalization of distinct domains of bRPGRIP1b in transiently transfected COS7 cells. Sample of cells transfected with the bRPGRIP1b construct (1.5 μg) tagged at the N- and C-terminal ends with EGFP- and HA-epitopes, respectively, led to the nuclear and cytoplasmic (A–C), and exclusive nuclear localization of EGFP- signal (D–I). Cells lacking EGFP-signal in the cytosolic compartment presented strong and diffused localization of this signal in the nucleoli (D–I; arrows). In the vast majority of cells, the HA-signal was found exclusively in the cytosolic compartment (D–F). Often there were cells without HA-signal and these presented EGFP-signal only in the nucleus/nucleolus (G–I). EGFP- (images in the left column) and HA-signals (images in the middle column) are shown, respectively, in green and red. Overlay pictures of these are shown in the right column. Non-transfected cells are depicted with a star. Scale bar: 10 μm.

The ΔE1279 mutation is resistant to physico-chemical stresses. Serial-dilution yeast growth assays in solid selective growth media at 22°C with the same reporter assay and constructs described in and in the presence of various treatments. Lowering the temperature normalized the growth rates between wild-type and ΔE1279 RID constructs but it did not affect D1114G (left). Treatment with sorbitol, heat-shock pulses and decrease of pH, increased selectively the growth rate of ΔE1279 and it had variable effects on the counterpart wild-type construct. The chemical chaperone, DMSO, led to an increase of the growth rates of wild-type and ΔE1279 constructs. None of the treatments elicited growth of D1114G.

The ΔE1279 and D1114G genetic lesions in RID of RPGRIP1 are gain- and loss-of-function mutations. (A) Serial-dilution yeast growth assays (30°C) in solid non-selective (left) and selective (right) growth media. None of the constructs exhibited toxicity as surveyed by the growth (and morphology) of yeast colonies in non-selective growth media. In selective media, ΔE1279 mutation exhibited stronger growth and faster growth rates, whereas D1114G failed to grow. (B) GAL4-responsive UAS-his3 reporter growth assay (30°C) in selective liquid growth media of yeast cells coexpressing wild-type RID and mutations therein with RHD of RPGR. The ΔE1279 and D1114G mutations cause, respectively, an increase and decrease of ~35 and 95% of the strength of the interaction between RID and RPGR. Values are the average of three independent experiments and are shown with standard deviation. (C) Western blot analysis of homogenates of ~10⁶ yeast cells coexpressing RPGR with the GAL4AD-fused RID constructs. All RID constructs were expressed at similar levels.

Quantitative analysis of the subcellular localization of N- and C-terminal domains of bRPGRIP1, bRPGRIP1b and mRPGRIP1b in transiently transfected COS7 cells. The percentage of transfected cells showing N-terminal fused EGFP- (A, C) and C-terminal tag-fused (HA/Flag) positive signals (B, D) in the nuclear and cytosolic subcellular compartments (A, B) and pattern of cytosolic distribution of these (C, D) was determined for bRPGRIP1, bRPGRIP1b and mRPGRIP1b (see diagram in ). Shown are the mean ± standard deviations of data from three experiments. A minimum of 100 transfected cells were counted for each experiment. The EGFP- (A) and HA-positive (B) signals of mRPGRIP1b were always in the cytoplasm of every cell scored. In contrast, the majority of cells expressing bRPGRIP1 and bRPGRIP1b presented EGFP-signal in the nucleus (A), while the HA-signal (B) was confined mostly either to the cytosolic compartment or completely absent from it. The N- (C) and C-terminal (D) signal distribution pattern in the cytosolic compartment of mRPGRIP1b were always punctuate [and partially localized to a subpopulation of lysosomes ()] in the cytoplasm of every cell scored. Conversely, EGFP- (C) and HA-positive (D) signal distribution pattern of bRPGRIP1 and bRPGRIP1b were predominantly reticular in the cytosolic compartment, albeit some cells presented also a punctuate distribution pattern.

LCA-linked mutations in RID cause aberrant subcellular localization of RPGRIP1 and domains thereof. Localization of the mutations in RPGRIP1 studied and of the RPGRIP1 antigenic domain of Ab22 are depicted in the diagram shown above the image panels. Cells transfected with the wild-type (A–C), ΔE1279 (D–I) and D1114G (J–L) and ΔRID (equivalent to human Q893X) (M–O) mutations in RID of bRPGRIP1 construct (1.5 μg) tagged at the N-terminal end with EGFP, cause abnormal molecular and subcellular phenotypes. The ΔE1279 (D–I) mutation in RID led to an increased localization of the SMC/CC domain to the nuclear compartment. The SMC/CC domain never colocalized with the EGFP-fused N-terminal domain and it was excluded from the nucleolus (arrow). Moreover, its nuclear distribution pattern (as gross punctuate foci) was distinct from the diffused nuclear distribution of EGFP-fused domain. The D1114G mutation (J–L) in RID completely abolished the immunoreactivity SMC/CC domain towards its cognate antibodies (Ab22/23) in every cell scored and EGFP was often excluded from the nucleolus. The ΔRID (equivalent to human Q893X) mutation (M–O) led to a strong increase of the EGFP-fused N-terminal domain in the nucleus (and decrease from the cytosol), where it was often observed as strong/gross puncta. EGFP (images in the left column) and SMC/CC signals (images in the middle column) are shown, respectively, in green and red. Overlay pictures of these are shown in the right column. Scale bar: 10 μm.

RPGRIP1 is proteolytically cleaved into a stable N-terminal ~7 kDa fragment and the C-terminal fragment is degraded. RPGRIP1 isoform constructs and antibodies used in the immunoblots are depicted above the panels. Western blot analysis of homogenates of non- (NT) and transient-transfected COS7 cells using antibodies directed against the N-terminal fused EGFP (A), SMC/CC (B), RID (C) and C-terminal HA tag (D). All immunoblots were overexposed for increased sensitivity to detection of proteolytic product intermediates, which are unstable and of very low abundance (including the unprocessed protein). The EGFP antibody detected an abundant EGFP-fused protein of ~34 kDa (EGFP–p7; open arrowhead) (EGFP molecular mass is 27 kDa), whereas the ~200 kDa unprocessed EGFP–RPGRIP1 (arrow) was of very low abundance (A). At least two minor EGFP-fused proteolytic product intermediates were observed (A; filled arrowheads). In contrast, antibodies against SMC/CC (B), RID (C) and C-terminal HA tag (D) detected predominantly the unprocessed EGFP–RPGRIP1 (arrow). The high-affinity Ab38 against RID detected multiple proteolytic product intermediates of minor abundance (C; filled arrowheads). (E, F) Immunoblots of homogenates of nuclear (E) and cytosolic (F) fractions of non- (NT) and transient-transfected COS7 cells with bRPGRIP1 and bRPGRIP1b using antibodies directed against the N-terminal fused EGFP (A). Both bRPGRIP1 and bRPGRIP1b generate an abundant EGFP-fused protein of ~34 kDa (EGFP–p7; open arrowhead) that is present in the nuclear and cytosolic fractions, whereas unprocessed bRPGRIP1s are comparatively of minor abundance. The nuclear and cytoplasmic markers, Nup62 and calnexin, respectively, were used as controls (blots in the lower panel).

Model of the role of RPGRIP1 in neuroretinal function and molecular pathogenesis of LCA/XlRP3. Schematic diagram of RPGRIP1 depicting the nuclear (translocation) domain (ND) identified in this work with the consensus nuclear localization signals (NLS) in human (h) and bovine (b). Normal and pathological (e.g. D1114G mutation) proteolytic sites are noted with filled and dashed arrows. The model depicts two independent roles of p175/RPGRIP1 in retinal function (for detailed information see discussion), one in photoreceptor neurons and another in inner (post-receptoral) neurons. In the photoreceptors, RPGRIP1 (p175) is sequestered in the rod outer segments and it is not subjected to proteolysis. This process may be pathogenic in photoreceptors upon genetic lesions in RPGR and/or RPGRIP1 (p175). In mature post-receptoral neurons, RPGRIP1 (p175) is under constitutive proteolysis (possibly owing to the lack of RPGR and/or other factors) leading to the production of a processed and stable nuclear factor, p7. The mutations, D1114G and ΔE1279, in RPGRIP1 (p175) have several antagonistic effects on RPGRIP1 function. Among these are the effects on the interaction with RPGR, presence of SMC/CC in the nuclear/cytosolic compartments and production of proteolytic intermediate and stable products (p17).

Subcellular compartmentalization of distinct domains of bRPGRIP1 in transiently transfected COS7 cells. (A) Diagram depicting the EGFP-fused RPGRIP1 constructs employed in this study (see also and ). bRPGRIP1 and bRPGRIP1b were also fused at the C-terminus with HA-tags (HA), whereas the mRPGRIP1b contained a FLAG tag (FL). Cells transfected with the bRPGRIP1 construct (1.5 μg) tagged at the N- and C-terminal ends with EGFP- and HA-epitopes, respectively, led to the nuclear and cytoplasmic (B–D, H–J), and exclusive nuclear localization of EGFP-signal (E–G, K–M). When present in the cytoplasm, the nuclear localization of EGFP was often diffused (B–D, H–J). Conversely, cells lacking EGFP-signal in the cytosolic compartment presented a strong and diffused localization of this signal in the nucleoli (E–G, K–M; arrows). In the cytoplasm, EGFP was found distributed in a reticular (B–D) and strongly punctuate (H–J) pattern, albeit the former predominated. In the vast majority of cells, the HA-signal was found exclusively in the cytosolic compartment, where it colocalized always with the EGFP-signal independently of its distribution pattern (B–D, H–J). EGFP- (images in the left column) and HA signals (images in the middle column) are shown, respectively, in green and red. Overlay pictures of these are shown in the right column. Scale bar: 10 μm. SMC/CC, α-helical coiled coil-hinge-coiled coil protein interaction motif of members of the structural maintenance of chromosomes (SMC) superfamily; C2, protein kinase C conserved region 2 and Ca²⁺-binding motif (C2); RID, RPGR-interacting domain.

Quantitative analysis of the subcellular localization of N-terminal and SMC/CC-domains of bRPGRIP1 with LCA-linked mutations and biochemical characterization of these in transiently transfected COS7 cells. Blind sample of cells transfected with the wild-type, ΔE1279 and D1114G and ΔRID (equivalent to human Q893X) mutations in the bRPGRIP1 construct described in were scored for the subcellular localization of EGFP and SMC/CC domains, and immunoreactivity of the latter. Shown are the mean ± standard deviations of data from three experiments. A minimum of 100 transfected cells were counted for each experiment. The D1114G mutation leads to a redistribution of the EGFP-fused N-terminal of bRPGRIP1 between the nuclear and cytosolic (N + C) compartments (A), almost no cells were observed with EGFP signal in the cytosolic compartment alone (A) and there were no SMC/CC-positive compartments and cells detected (B). The ΔE1279 mutation causes an increased localization of the EGFP-signal in the cytosolic (C) compartment alone (A) and it leads to an increase of SMC/CC in the nucleus (N + C, N) that was accompanied by a decrease in the cytosol (C) alone (B). The ΔRID mutation leads to a strong increase (retention) of EGFP in the nuclear (N) compartment alone (A) and this was accompanied by an increase of SMC/CC-positive nuclear compartments (B). (C) An immunoblot of cell homogenates with EGFP antibody of non (NT, lane 1) and -transient transfected cells with EGFP alone (lane 2), EGFP–bRPGRIP1 (lane 3), EGFP–bRPGRIP1 with D1114G (lane 4), ΔE1279 (lane 5) and ΔRID (lane 6) mutations. Both the wild-type and all mutant constructs generate a N-terminal fragment of ~34 kDa (p7 fused to EGFP). However, the D1114G mutation causes the production of an aberrant, abundant and stable EGFP-fused proteolytic product of ~44 kDa (p17 fused to EGFP). In comparison to the wild-type protein (lane 3, filled arrow), the ΔE1279 mutation decreases the electrophoretic mobility of the unprocessed bRPGRIP1 (lane 5, open arrow).