PMC

Ubp64 interacts genetically with Ttk. (A and B) Scanning electron micrographs of adult eyes of WT flies (A) and GMR-Gal4 UAS-Ttk69/CyO flies (GMR>Ttk) (B) that ectopically express TTK under the control of the GMR enhancer. TTK misexpression during development causes a distorted eye morphology characterized by disorganized facets and loss of bristles. The TTK misexpression phenotype is suppressed when the UBP64 protein level is reduced in a ubp64^Δ1 background (GMR-Gal4 UAS-Ttk69/+ ubp64^Δ1/+, abbreviated as GMR>Ttk/ubp64^Δ1) (C) but not in a Rev background (GMR-Gal4 UAS-Ttk69/+ Rev/+, abbreviated as GMR>Ttk/Rev) (D). (E) Ectopic expression of both TTK and UBP64 in GMR-Gal4 UAS-Ttk69/+ UAS-Ubp64/+ flies (GMR>Ttk/GMR>Ubp64) dramatically disrupts eye formation. (F) Combined overexpression of TTK and the enzymatically inactive UBP64^C405A in GMR-Gal4/+ UAS-Ubp64^C405A/+ GMR-Gal4 UAS-Ttk69/+ flies (GMR>Ttk/GMR>Ubp64^C405A) did not affect the eye phenotype.

UBP64 inhibits photoreceptor differentiation. (A) To identify photoreceptor cells, eye discs from WT pupae were immunostained with antibodies directed against the neuronal marker ELAV. Note that although WT ommatidia harbor eight photoreceptor cells, only seven are visualized because the R8 photoreceptor lies below the selected focal plane. A higher magnification of a representative ommatidium is shown at the bottom. (B) Homozygous ubp64^Δ1/ubp64^Δ1 ommatidia are disorganized and frequently contain supranumerical photoreceptors. (C) Rev retina is similar to WT retina. (D) Ectopic expression of UBP64 in GMR-Gal4/+ UAS-Ubp64/+ pupal retinae (GMR>Ubp64) disrupts the regular arrangement of ommatidia and inhibits photoreceptor cell formation, leading to fewer than the normal complement of eight photoreceptors. (E and F) Ectopic expression of TTK69 in GMR-Gal4 UAS-Ttk69/Rev pupae (GMR>Ttk69/Rev) strongly disrupts the photoreceptor pattern (E), which is suppressed by the reduced UBP64 levels in GMR-Gal4 UAS-Ttk69/+ ubp64^Δ1/+ (GMR>Ttk69/ubp64^Δ1) pupae (F). (G and H) Ectopic expression of both UBP64 and TTK69 in GMR-Gal4 UAS-Ttk69/+ UAS-Ubp64/+ pupae (GMR>Ttk69/GMR>Ubp64) enhances this phenotype further (G), whereas the expression of catalytically dead Ubp64^C405A in GMR-Gal4 UAS-Ttk69/+ UAS-Ubp64^C405A/+ pupae (GMR>Ttk69/GMR>Ubp64^C405A) does not enhance the effects of TTK69 overexpression (H).

UBP64 promotes cone cell formation. Eye discs isolated from 40-h-old pupae were analyzed and visualized by indirect immunofluorescence using antibodies directed against the cone cell-specific marker protein CUT. (A) Well-organized, repetitive pattern of ommatidia in WT pupal retina. Each ommatidium contains four CUT-expressing cone cells, as illustrated by the higher magnification of a representative ommatidium (bottom). (B) Ommatidia in homozygous ubp64^Δ1/ubp64^Δ1 mutant (ubp64^Δ1) eye discs are less well organized and frequently contain fewer than four cone cells. (C) Rev retinae are similar to WT retinae. (D) Ectopic expression of UBP64 in GMR-Gal4/+ UAS-Ubp64/+ (abbreviated as GMR>Ubp64) pupal retinae disrupts the regular arrangement of ommatidia and leads to the appearance of extra cone cells. (E) Ectopic expression of TTK69 in GMR-Gal4 UAS-Ttk69/Rev pupae (GMR>Ttk69/Rev) strongly disrupts the ommatidial pattern. (F) A reduction of the UBP64 level in GMR-Gal4 UAS-Ttk69/+ ubp64^Δ1/+ (GMR>Ttk69/ubp64^Δ1) pupal retinae suppresses the TTK69 misexpression phenotype. Note that ubp64^Δ1 and Rev have a common genetic background. (G) In contrast, the ectopic expression of both UBP64 and TTK69 in GMR-Gal4 UAS-Ttk69/+ UAS-Ubp64/+ (GMR>Ttk69/GMR>Ubp64) pupae enhances the phenotype further. (H) Ectopic expression of catalytically dead Ubp64^C405A in GMR-Gal4 UAS-Ttk69/+ UAS-Ubp64^C405A/+ pupae (GMR>Ttk69/GMR>Ubp64^C405A) does not enhance the effects of TTK69 overexpression.

UBP64 stabilizes transcription factor TTK. (A) UBP64 and associated proteins immunopurified from 1 ml of Drosophila embryo nuclear extract (concentration of 10 mg/ml) using affinity-purified antibodies coupled to protein A-Sepharose CL-4B beads. Following extensive washes with HEMG buffer containing 600 mM KCl and 0.1% NP-40, bound proteins were eluted with 100 mM glycine (pH 2.5), resolved by SDS-PAGE, and visualized by Coomassie staining. Approximately 0.03% of the input material was loaded. Mass spectrometric analysis was used to identify UBP64 and associated proteins not present in the mock purification (see Table ). Protein identities are indicated. MW, molecular weight (in thousands). (B) UBP64, TTK88, and TTK69 are associated in crude embryo nuclear extract. Nuclear extract (input) was incubated with protein A-Sepharose beads coated with anti-UBP64 antibodies. For mock immunoprecipitation (IP), protein A-Sepharose beads were directly added to the nuclear extract. After extensive washes with a buffer containing 300 mM KCl and 0.1% NP-40, bound proteins were resolved by SDS-PAGE and analyzed by Western immunoblotting with antibodies directed against either UBP64, TTK88, or TTK69. Ten percent of the input material was loaded. TTK69 levels are strongly reduced in ubp64^Δ1 flies, as revealed by Western immunoblot analysis of protein extracts prepared from either WT or ubp64^Δ1 eye discs. Anti-Moira (α-MOR) immunoblotting was performed as a loading control.

UBP64 deubiquitylates TTK to counteract Sina-directed degradation. (A) Recapitulation of SINA-dependent TTK polyubiquitylation. Plasmids expressing HA-tagged TTK69, SINA, and His₆-ubiquitin were cotransfected into H1299 cells in the indicated combinations. Proteasome-mediated degradation was inhibited by the addition of the drug MG132. After extract preparation, ubiquitylated proteins were purified under denaturing conditions by Ni²⁺-NTA chromatography, resolved by SDS-PAGE, and visualized by immunoblotting with antibodies directed against TTK69. (B) The relevant Coomassie-stained protein bands corresponding to the blot in A were excised and subjected to mass spectrometric analysis. The cartoon depicts the lysine residues that were covalently attached to the LRGG or GG ubiquitin remnants via an isopeptide bond. The sequences of the tryptic peptides and the ubiquitylated residue (red) are listed along with their mascot scores. (C) UBP64 stabilizes TTK69. A vector expressing HA-tagged TTK69 was cotransfected into H1299 with Sina and Ubp64 in the indicated combinations. In the absence of a proteasome inhibitor, the expression of Sina led to strongly reduced TTK69 levels. TTK69 degradation was effectively counteracted by the coexpression of UBP64. TTK69 was detected using anti-HA antibodies. Histone H3 and an anti-HA cross-reacting protein (*) serve as loading controls. (D) Recombinant GST-tagged UBP64 and UBP64^C405A were purified from E. coli cell extracts by glutathione-Sepharose (fast flow) chromatography, resolved by SDS-PAGE, and visualized by Coomassie staining. MW, molecular weight (in thousands). (E) UBP64 deubiquitylates TTK. Ni²⁺-NTA-purified Ub-TTK69 was incubated in the absence or presence of either UBP64 or UBP64^C405A, resolved by SDS-PAGE, and analyzed by Western immunoblotting using anti-HA antibodies.

ubp64 mutants have defective eye development. (A to C) UBP64 expression was monitored by whole-mount immunostaining of WT (A), ubp64^Δ1 (ubp64^Δ1/ubp64^Δ1) (B), and revertant (Rev) (C) embryos using affinity-purified antibodies directed against UBP64. UBP64 is widely expressed and present in the cytoplasm and nucleus. UBP64 levels are severely reduced in ubp64^Δ1 embryos compared to WT and Rev embryos. (D to F) Scanning electron micrographs of adult eyes of WT (D), ubp64^Δ1 (E), and Rev (F) flies. An enlarged zoom image is shown at the bottom of each panel. In contrast to WT or Rev eyes, ubp64^Δ1 eyes have disorganized facets and bristles, which gives the external surface a rough appearance. In addition, there are supernumerary bristles, three examples of which are indicated with arrows. (G) Western immunoblotting analysis of UBP64 protein levels in extracts prepared from isolated pupal retinae and resolved by SDS-PAGE. Three and nine microliters of extract prepared from 10 retinae dissolved in 50 μl sample buffer was loaded (indicated with a triangle). Detection of histone H3 served as a loading control. (H to J) UBP64 expression was monitored in the pupal retinae of WT (H), ubp64^Δ1 (I), or Rev (J) animals. UBP64 is ubiquitously expressed in both the cytoplasm and nucleus of retina cells, but its expression level is severely diminished in ubp64^Δ1 animals. (K and L) Scanning electron micrographs of adult eyes of GMR-Gal4/+ UAS-Ubp64/+ flies (abbreviated as GMR>Ubp64) (K) and GMR-Gal4/+ UAS-Ubp64^C405A/+ (GMR>Ubp64^C405A) (L) flies. The ectopic overexpression of UBP64 in precursor cells posterior to the morphogenetic furrow, directed by the GMR enhancer, strongly disrupts eye development and loss of bristles. In contrast, the overexpression of the catalytically dead UBP64^C405A did not affect eye development. (M) Immunoblot analysis of extracts prepared from dissected eye discs confirmed the comparable levels of WT and mutant UBP64. Histone H3 serves as a loading control.