Figure 5.
Gonads of young flies possess more abundant and more active proteasomes than somatic tissues. A1–A5) Representative immunohistochemical staining of whole female fly sections with an anti-20S-α proteasome subunit antibody. The antigen was distributed in head (H), thorax (T), and abdomen (A) tissues (A1). More intense staining was seen in the ovarian tissue (ov; A1, dashed line), in the neural tissues of the head [e.g., the eye (E) and the brain (B); A2], and in muscle (M; A3). In the ovarian follicles (F; A4), the 20S-α proteasome subunit was distributed in the nurse cells (NC), the oocyte (OC), and the follicular epithelium (FC) (A5). In all cell types, (including muscle cells; A3) both cytoplasmic and nuclear (arrows) staining was seen. Scale bars = 50 μm. B) Comparative RT-PCR analyses of mRNA expression levels of indicated proteasome subunits in somatic (SO) tissues and gonads (OV, ovary; SP, permathecae) of female and male flies. C) Immunoblotting analysis of proteasome subunit expression levels in gonads and somatic tissues of female and male flies. D) Immunoprecipitation (IP) of 20S-α and β5 proteasome subunit interaction (indicating assembled proteasomes) in lysates from Drosophila gonads and somatic tissues. Immunoprecipitation was performed with the anti-20S-α antibody, and immunoprecipitates were probed by immunoblotting (IB) with anti-20S-α and anti-β5 antibodies. E) Relative activity (%) of 26S (top panel) and 20S (bottom panel) proteasomes in somatic tissues and gonads of Drosophila. In nearly all cases, significantly higher proteasome peptidase activity was found in the gonads. F1, F2) Immunoprecipitation of the 20S-α proteasome subunit followed by immunoblotting analysis of 20S-α and β5 subunits. A minimal amount of the anti-20S-α antibody was used to achieve the immunoprecipitation of equal amounts of proteasomes from different tissues. F1) Indeed, although the unbound ovarian fraction contained (compared to somatic tissues) higher amounts of proteasomes, we detected similar amounts of assembled proteasomes in the eluate. F2) Relative activity (%) of 26S proteasome in the unbound fraction and immunoprecipitated proteasomes in the female soma and ovaries. Note higher endogenous proteasome catalytic activity in the immunoprecipitated ovarian proteasomes. All assays were done in 10- to 13-d-old flies. GAPDH and rp49 were used as references for equal total protein and RNA input, respectively. Bars represent means ± sd; n = 2. *P < 0.05, **P < 0.01.