Molecular architecture and spatial organisation of proteasomes in the human sperm nucleus

Proteasomes are proteolytic machines essential for proteostasis and genome integrity. They consist of a 20S core particle capped by regulatory complexes that confer substrate selectivity (Voges, 1999). Despite evidence that proteasome function is necessary for successful spermatogenesis and fertilisation (Sutovsky, 2011; Xiong et al., 2022), the presence, composition, and role of proteasomes within the sperm nucleus have remained a subject of debate. Here, we used in situ electron cryo-tomography (cryo-ET) in human sperm cells to elucidate the molecular architecture of nuclear proteasomes, which cluster in DNA-free, nuclear cavities within the sperm nucleus. Subtomogram averaging revealed that the main population of proteasomes consists of 20S core particles, with a smaller fraction of 20S capped by PA200 activator, solved at sub-nanometre resolution. Subsequent single-particle cryo-EM analysis of purified native human sperm proteasomes further elucidated atomic features of the essential testis-specific subunit 4s, showing the presence of a unique splice variant. Notably, our reconstruction resolved a native peptide in the catalytic {beta}2 subunit, providing novel insights into the proteolysis mechanism and PA200-mediated enhancement of trypsin activity. Finally, we show nuclear enrichment of proteasomes during sperm cell differentiation in human testis tissue, with 20S and PA200 clustering following meiosis, at the spermatid stage. Our findings shed light on the unique organisation and compositional diversity of nuclear proteasomes in human sperm cells, disclosing novel molecular insights into their catalytic function.