Summary The Ubiquitin-Proteasome System catalyzes the degradation of intracellular proteins. Although ubiquitination of proteins determines their stabilities, there is growing evidence that proteasome function is also regulated. We report the functional characterization of a conserved proteasomal regulatory complex. We identified DmPI31 as a binding partner of the F-box protein Nutcracker, a component of an SCF ubiquitin ligase (E3) required for caspase activation during sperm differentiation in Drosophila. DmPI31 binds Nutcracker via a conserved mechanism that is also used by mammalian FBXO7 and PI31. Nutcracker promotes DmPI31 stability, which is necessary for caspase activation, proteasome function and sperm differentiation. DmPI31 can activate 26S proteasomes in vitro, and increasing DmPI31 levels suppresses defects caused by diminished proteasome activity in vivo. Furthermore, loss of DmPI31 function causes lethality, cell-cycle abnormalities and defects in protein degradation, demonstrating that DmPI31 is physiologically required for normal proteasome activity.

The testis is a peculiar tissue in many respects. It shows patterns of rapid gene evolution and provides a hotspot for the origination of genetic novelties such as de novo genes, duplications and mutations. To investigate the expression patterns of genetic novelties across cell types, we performed single-cell RNA-sequencing of adult Drosophila testis. We found that new genes were expressed in various cell types, the patterns of which may be influenced by their mode of origination. In particular, lineage-specific de novo genes are commonly expressed in early spermatocytes, while young duplicated genes are often bimodally expressed. Analysis of germline substitutions suggests that spermatogenesis is a highly reparative process, with the mutational load of germ cells decreasing as spermatogenesis progresses. By elucidating the distribution of genetic novelties across spermatogenesis, this study provides a deeper understanding of how the testis maintains its core reproductive function while being a hotbed of evolutionary innovation.

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Here we report the characterization of an eps15 homology (EH) domain containing protein designated EHD3. EHD3 was mapped to human chromosome 2p22-23, while the murine Ehd3 homolog was mapped to chromosome 17p21. Both the human and the mouse genes contain a polymorphic (CA) repeat in their 3ƒUTR. One 3.6-kb Ehd3 transcript was mainly detected in adult mouse brain and kidney and at day 7 of mouse development. On the other hand, human tissues exhibited two, 4.2-and 3.6-kb, EHD3 RNA species. They were predominantly expressed in heart, brain, placenta, liver, kidney and ovary. EHD3, expressed as a green fluorescent fusion protein was localized to endocytic vesicles and to microtubule-dependent, membrane tubules. There was a clear colocalization of EHD3-positive structures and transferrincontaining recycling vesicles, implying that EHD3 resides within the endocytic recycling compartment. Shuffling the N-terminal domain of EHD1 (previously shown to reside in the transferrin-containing, endocytic recycling compartment) with that of EHD3 resulted in a chimeric EHD protein that was localized mainly to tubules instead of the endocytic vesicles, implicating the N-terminal domain as responsible for the tubular localization of EHD3. Mutant EHD3 forms, missing the N-terminal or the C-terminal domains, lost their tubular localization. Results of two-hybrid analyses indicated that EHD1 and EHD3 interact with each other. In addition, EHD1 and EHD3 could be coimmunoprecipitated from cellular extracts, confirming the interaction implied by two-hybrid analysis. Moreover, coexpression of EHD1 and EHD3 resulted in their colocalization in microtubule-dependent tubules as well as in punctate forms. Based on its specific intracellular localization and its interaction with EHD1, we postulate that EHD3 localizes on endocytic tubular and vesicular structures and regulates their microtubule-dependent movement.