Identification and validation of TULP3 protein–protein interactions.A, silver stained gel showing proteins coimmunoprecipitated alongside mock-FLAG or FLAG-TULP3 from 293T cells. The band corresponding to FLAG-TULP3 is indicated by a red arrow. B, bar graph ranking the top 35 high-confidence hits in (A) as identified by mass spectrometry. C, STRING diagram outlining the interconnectedness of TULP3 interactors identified in (B). Western blots showing coimmunoprecipitation of FLAG-TULP3 with (D) SIRT1, (E) RAD18, and (F) PP6R3 in 293T cells.

Acetylation of TULP3 by p300 increases its protein abundance in cells. A, western blot showing TULP3 acetylation levels in 293T cells in the presence of either empty pcDNA 3.1(+), Myc-p300, FLAG-PCAF, or FLAG-GCN5 following immunoprecipitation. Cells were harvested 48 h posttransfection. B, semiquantitative LC-MS/MS-based comparison of TULP3 acetylation corresponding to the conditions in (A). C, western blot showing total levels of endogenous TULP3 in HeLa cells following transfection with empty pcDNA 3.1(+) or a plasmid encoding HA-p300. Cells were harvested 48 h posttransfection. D, mRNA levels of endogenous TULP3 corresponding to samples in (C) examined using quantitative real-time PCR; n = 4 technical replicates, Mean ± S.D. shown. E, western blot of p300 and TULP3 protein levels following stable transduction of p300 shRNAs into HeLa cells. F, immunoblot of stably expressed FLAG-TULP3 protein in 293T cells following treatment with either DMSO, 30 μM C646, or 50 μM anacardic acid for 24 h. G, mRNA levels of FLAG-TULP3 corresponding to samples in (F) analyzed using quantitative real-time PCR; n = 4 technical replicates, Mean ± S.D. shown. H, acetylation levels of FLAG-TULP3 in stably expressing 293T cells following treatment with DMSO or 30 μM C646 for 9 h.

Modification of K316, K320, and K389 each influences TULP3 stability.A, schematic outlining key functional domains in TULP3 and the location of acetylation sites. IFT-A ID denotes Intraflagellar Transport Complex A interacting domain. Western blots representing cycloheximide pulse-chase experiments for stably expressed (B) FLAG-TULP3 wild-type protein or (C) K316Q, (D) K316R, (E) K389Q, (F) K389R, (G) K316Q/K389Q, (H) K316R/K389R mutants in 293T cells. I, plot corresponding to experiments in (B–H) quantifying relative TULP3 protein levels versus time; n = 3 biological replicates, Mean ± S.D. shown. J, protein levels of FLAG-TULP3, FLAG-TULP3 K316Q/K389Q, and K316R/K389R mutants stably expressed in 293T cells following treatment with DMSO or 30 μM C646 for 9 h.

HDAC1 deacetylates TULP3 and reduces its protein levels.A, western blot showing acetylation and total protein levels of stably expressed FLAG-TULP3 in 293T cells treated with DMSO, 1 μM TSA, 1 μM MC1568, 1 μM HDAC Inhibitor XXIV, or 10 μM MS-275 for 16 h. B, acetylation and total protein levels of stably expressed FLAG-TULP3 in 293T cells transfected with empty pcDNA 3.1(+) or a plasmid encoding HA-HDAC1. Cells were harvested 48 h posttransfection. C, western blot showing endogenous levels of TULP3 in Hela cells transfected with either empty pcDNA 3.1(+) or a plasmid encoding FLAG-HDAC1. Cells were harvested 48 h posttransfection. D, mRNA levels of endogenous TULP3 corresponding to treatments in (C) analyzed by qRT-PCR; n = 4 technical replicates, Mean ± S.D. shown. E, western blot of HDAC1 and TULP3 protein levels following stable transduction of HDAC1 shRNAs into HeLa cells. Western blots showing coimmunoprecipitation of F FLAG-TULP3 with HDAC1 or G HA-HDAC1 with FLAG-TULP3 in 293T cells.

TULP3 protein stability is regulated by polyubiquitination and by Cullin-3. A, western blot showing TULP3 levels in HeLa cells treated with 10 μM MG-132 for the time intervals indicated. B, TULP3 protein levels in the absence or presence of 10 μM MG-132 and transfected with a plasmid encoding HA-p300 as indicated. Cells were harvested 48 h posttransfection following 9 h of MG-132 treatment. C, immunoblot analysis of ubiquitination levels on FLAG-TULP3 immunoprecipitated from 293T cells treated with MG-132 and transfected with plasmids encoding HA-Ubiquitin or Myc-p300 as indicated. Cells were harvested 48 h posttransfection following 9 h of MG-132 treatment. D, western blot showing ubiquitination levels on FLAG-TULP3 immunoprecipitated from 293T cells treated with MG-132 and transfected with plasmids encoding HA-Ubiquitin or Myc-HDAC1 as indicated. Cells were harvested 48 h posttransfection following 9 h of MG-132 treatment. E, schematic outlining key functional domains in TULP3 and the location of ubiquitination sites identified by mass spectrometry. IFT-A ID denotes Intraflagellar Transport Complex A interacting domain. F, western blot showing coimmunoprecipitation of FLAG-TULP3 with Cullin-3 in 293T cells. G, immunoblot showing levels of FLAG-TULP3 protein in 293T cells in the absence or presence of C646 following transfection with a pool of siRNAs targeting Cullin-3 or a scrambled control. Cells were collected 72 h posttransfection; 30 μM C646 was added to cells 48 h posttransfection as indicated.

An acetylation switch actively regulates TULP3 levels in zebrafish during development.A, immunoblot showing TULP3 protein levels in zebrafish embryo lysates harvested 10 h postfertilization following treatment with DMSO or 3 μM C646 for 10 h. B, representative photos of zebrafish embryos 24 h postfertilization treated with either DMSO or 3 or 5 μM C646 for 24 h. C, graph quantifying the frequency of the phenotypes observed in panel (B); n = 120, 123 or 123 for 0, 3, and 5 μM C646 groups respectively. Mean ± S.D. shown. ∗∗∗∗ denotes p < 0.0001 (t test). D, western blot showing levels of TULP in zebrafish embryos 24 h postfertilization in the absence or presence of 8 ng TULP3 morpholino. E, representative photos of zebrafish embryos 24 h postfertilization displaying normal, delayed, mild apoptosis, and severe apoptosis phenotypes. The black and red arrows indicate apoptosis in the hindbrain and forebrain, respectively. F, graph quantifying the frequency of the phenotypes observed in panel (E); n = 271, 248, 317, or 292 for control, morpholino only, morpholino + WT, or morpholino + 2Q mutant, respectively. Mean ± S.D. shown. ∗ denotes p < 0.05, ∗∗ denotes p < 0.01, and n.s. indicates nonsignificance (t test). G, levels of TULP3 protein in morpholino-injected zebrafish rescued with either wild-type TULP3 mRNA or K316Q/K389Q TULP3 mRNA. Lysates were harvested 8 h postfertilization. H, immunoblot showing protein levels of TULP3 and p300 in zebrafish embryo lysates harvested at 4, 8, 24, and 32 h postfertilization.

p300 and HDAC1 influence the stability of multiple tubby-like proteins.A, sequence alignment of the four human TULP proteins (TULP1-4) performed using ClustalW software. Identical, similar, and nonsimilar residues are color coded as indicated. Key acetylation sites are indicated with arrows. B, western blot showing TULP1, TULP2, and TULP4 protein levels in 293T cells transfected with either a control plasmid or a plasmid encoding HA-p300. Cells were harvested 48 h posttransfection. C, western blot showing levels of TULP1, TULP2, and TULP4 in 293T following 24 h treatment with either DMSO or 30 μM C646. D, western blot of p300, TULP1, TULP2, and TULP4 protein levels following stable transduction of p300 shRNAs into 293T cells. E, western blot showing TULP1, TULP2, and TULP4 protein levels in 293T cells transfected with either a control plasmid or a plasmid encoding Myc-HDAC1. Cells were harvested 48 h posttransfection. F, immunoblot showing p300, TULP1, TULP2, and TULP4 protein levels following stable transduction of HDAC1 shRNAs into 293T cells. G, western blot showing levels of TULP1, TULP2, and TULP4 in 293T cells following 9 h treatment with either DMSO or 10 μM MG-132.