Fig 2: Exogenous ?-tubulin 2 locates to centrosomes and interacts with GCPs.(A) Localization of FLAG-tagged ?-tubulins. Human U2OS cells expressing mouse ?-tubulin 1 (a, Tubg1-FLAG), mouse ?-tubulin 2 (b, Tubg2-FLAG), human ?-tubulin 1 (c, TUBG1-FLAG) and human ?-tubulin 2 (d, TUBG2-FLAG) were stained for FLAG (red) and pericentrin (green). DNA was stained with DAPI (blue). Arrows denote positions of MTOCs where FLAG-tagged ?-tubulins co-localize with pericentrin. Final images were made by maximum intensity projection of 3 deconvolved z-sections spaced at 0.25 µm. Scale bar 10 µm. (B) Coimmunoprecipitation of mouse ?-tubulins with GCP2 and GCP4 proteins. Extracts from HEK cells expressing FLAG-tagged ?-tubulin 1 (Tubg1-FLAG), ?-tubulin 2 (Tubg2-FLAG) or control mouse Fyn (Fyn-FLAG) were immunoprecipitated with antibodies to FLAG or GCP2, and blots were probed with antibodies to FLAG, GCP2, GCP4 and ?-tubulin (?-Tb). Extracts (1), immunoprecipitated proteins (2), protein A without antibodies incubated with extracts (3), immobilized antibodies not incubated with extracts (4). Arrowheads indicate the positions of exogenous ?-tubulins.

Fig 3: Profilin interacts with ?-TuRC.(A, B, C) Extracts of B16 cells were precipitated with immobilized Abs to profilin (A), ?-tubulin (B) or GCP2 (C), followed by Western blotting and probing with Abs against profilin (Pfn), ?-tubulin (?-Tb), GCP2, GCP4, and calmodulin (negative control) as indicated (C). In each blot (A, B, C), the sample lanes denoted 1–4 show: total cell extract, gel load (1); no cell extract (2); precipitated proteins (3); and Ab-free carrier (4). Note reciprocal precipitation in all three cases. (D) Extracts of B16 control (1, 4, 6) and profilin knock-out KO27 (2, 5, 7) cells were precipitated with immobilized Abs to profilin, followed by Western blotting and probing with Abs against profilin (Pfn), ?-tubulin (?-Tb), GCP2, GCP4, actin, and calmodulin (negative control) as indicated. The sample lanes denoted 1–7 show: total cell extract, gel load (1, 2); no cell extract (3); precipitated proteins (4–5); and Ab-free carrier (6–7). Note co-precipitation of ?-tubulin, GCP2, GCP4, and actin with profilin Ab occurs in B16 cells solely. (B, D) Asterisks (*) in panels (B) and (D), lanes 2 and 3, and 3–5, respectively, denote cut off bands reflecting precipitating Abs.

Fig 4: UFL1 and C53 interact with ?TuRC proteins. (A) Immunoprecipitation experiments. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized Abs specific to UFL1301–389, C53, ?-tubulin (?-Tb), or GCP2. The blots were probed with Abs to UFL1, C53, ?-tubulin (?-Tb), GCP2, GCP4, or calcineurin (Calcin.; negative control). Load (lane 1), immobilized Abs without cell extracts (lane 2), precipitated proteins (lane 3), and Ab-free carriers incubated with cell extracts (lane 4). (B) Isotype controls. Extracts from the membranous fraction (P2) of U2OS cells were precipitated with immobilized rabbit Ab to myosin or mouse mAb to MAP2 (IgG2b). Blots were probed with Abs to UFL1, C53, ?-tubulin (?-Tb), GCP2, or GCP4. Load (lane 1), immobilized Abs not incubated with cell extracts (lane 2), precipitated proteins (lane 3), and carriers without Abs incubated with cell extracts (lane 4). (C) The size distribution of UFL1, C53, ?-tubulin (?-Tb), GCP2, and actin in U2OS whole-cell extracts fractionated on the Superose 6 column. The calibration standards (in kDa) are indicated on the top. The numbers at the bottom denote individual fractions. (D) Pooled fractions (Nos. 15–16) and (Nos. 23–24) from fractionation shown in panel (C) were precipitated with Ab to ?-tubulin. The blots were probed with Abs to UFL1, C53, and ?-tubulin (?-Tb). Load (lane 1), immobilized Abs without cell extracts (lane 2), precipitated proteins (lane 3), and Ab-free carriers incubated with cell extracts (lane 4).

Fig 5: GCP4 inhibits autophagy by competing with ATG3 to interact with ATG7. a, b Co-immunoprecipitation analysis showed that GCP4 interacted with ATG7 in HEK293T cells. The 293T cells were transiently transfected with both pMyc-ATG7 and p3xFlag-TUBGCP4, and after transfection for 48 h, the whole cell lysates were extracted for co-immunoprecipitation with anti-MYC (a) or anti-FLAG (b). Anti-FLAG (a) or anti-MYC (b) antibody was used for Western blotting. c Schematic diagram of the ATG7 domains. NTD, N-terminal domain; CTD, C-terminal domain; AD, adenylation domain; ECTD, extreme C-terminal domain. d Co-immunoprecipitation analysis showed that GCP4 interacted with the NTD of ATG7. p3xFlag-TUBGCP4 was co-transfected with pMyc-ATG7-NTD, pMyc-ATG7-CTD into 293T cells. For co-immunoprecipitation, the lysates were immunoprecipitated with the anti-MYC antibody, followed by immunoblotting with the anti-FLAG antibody. The result showed that FLAG-GCP4 can interact with MYC-ATG7-NTD. e GCP4 can interact with ATG7 in vivo. Retina cell lysates were extracted for immunoprecipitation with anti-ATG7 (Rabbit IgG for control) followed by immunoblotting with antibody against GCP4. f, g GST-pulldown assay showed GCP4 interaction with the N-terminal of ATG7. Coomassie gel showed expression of His-GCP4, GST-ATG7, GST-ATG7-N, GST-ATG7-C, and GST in supernatant of E. coli culture (f). His-tagged GCP4 was incubated with GST-ATG7, GST-ATG7-N, GST-ATG7-C and GST respectively. Proteins pulled down with glutathione-agarose were subjected to SDS-PAGE followed by immunoblotting with anti-GCP4 antibody (g). h Co-localization of ATG7 and GCP4 in OS, IS, OPL, IPL, and GCL of retinas by immunofluorescence analysis using anti-GCP4 (second antibody, Alexa Fluor 594-conjugated-goat anti-Rabbit) and Alex488-labeled anti-ATG7. The nuclei were stained with Hoechst reagent. RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 50 µm. i GCP4 competes with ATG3 to interact with ATG7. pMyc-ATG7 was co-transfected with p3xFlag-ATG3 (1 µg) or p3xFlag-TUBGCP4 (1 µg) and an increasing amount (0, 0.3, 0.6, and 1.2 µg) of p3xFlag-TUBGCP4 (left panel) or p3xFlag-ATG3 (right panel). The cell lysates were immunoprecipitated with the anti-MYC antibody, followed by immunoblotting with the anti-FLAG antibody. The whole cell lysates were examined by western blotting using the anti-FLAG or anti-MYC antibody (input). j A model of GCP4 inhibiting autophagy through competing with ATG3 to interact with ATG7. The free ATG7, which is a key autophagy protein to initiate the formation of phagophores, can interact with either ATG3 or GCP4 through its NTD. When Tubgcp4 is knocked out, ATG3 interacts with ATG7 to promote lipidation of LC3B and autophagy. In wild-type cells, GCP4 can compete ATG3 to interact with ATG7, which inhibits lipidation of LC3B and autophagy. Thus, GCP4 balances functions of ATG3 in autophagy regulation