Fig. 6.
Cog7 interacts with GOLPH3 and is required for proper localization of GOLPH3 protein to Golgi membranes. (A) Frequency of spermatids, containing 1, 2, 3, 4 or more than 4 nuclei per mitochondrial derivative, in testes of control males (Df(2L)Exel7010/+) and males that are either hemizygous for GOLPH3 (GOLPH3z2217/Df(2L)Exel7010), or heterozygous for Cog7 and hemizygous for GOLPH3 (GOLPH3z2217/Df(2L)Exel7010; Cog7z4495/+). (B) Cog7 mutants display a decreased amount of GOLPH3 protein at the Golgi membranes. Interphase spermatocytes from wild type and Cog7z4495/Df(3R)BSC861 (Cog7) mutants were stained for GOLPH3, the Golgi marker Lava lamp (Lva) and DNA. GOLPH3 levels in the Golgi were quantified as mean fluorescence intensity of GOLPH3 in Lva-positive regions (Lva+). In total, we examined 112 Golgi from Cog7z4495/Df(3R)BSC861 (Cog7), and 105 Golgi in wild-type interphase spermatocytes. The cells examined for Golgi analysis were randomly selected from three independent experiments. (C) PLA to visualize GFP–Cog7/GOLPH3 interaction in fixed spermatocytes. PLA with monoclonal antibodies against GFP and GOLPH3 (rabbit anti-GOLPH3 G49139/77) was used to test the interaction in interphase spermatocytes stained for DNA. Zoomed panel shows co-localization of the proteins at the Golgi. Negative control experiments were performed with antibodies against GFP and anillin. Scale bars: 10 µm. (D) Average number (±s.e.m.) of PLA dots per cell in interphase spermatocytes. Quantification of the number of PLA signals per cell was obtained as described in the Materials and Methods. (E) Oregon-R GOLPH3z2217 flies display regular wings, but GOLPH3z2217;Cog7z4495/+ exhibit ‘held out wings’ posture. In addition, the wings of GOLPH3z22177; Cog7z4495/+ appear severely swollen and crumpled. (F) Loss of Cog7 impairs the interaction of GOLPH3 with the coatomer protein α-COP. Protein extracts from fly heads were immunoprecipitated with antibodies against Drosophila GOLPH3 (rabbit G49139/77) and blotted with mouse anti-GOLPH3 S11047/1/56, guinea pig anti-α-COP, mouse anti-Rab1 S12085a antibodies. Preimmune serum (PI; G49139/1, from the same animal before the immunization), was used as control. 2% of the total lysate and one-third of the immunoprecipitates were loaded and probed with the indicated antibody. Molecular mass is given in kilodaltons. The Co-IP experiment was performed three times with identical results. Error bars indicate s.e.m. ***P<0.0001 (unpaired Student's t-test).