Repurposing of the assembly chaperone PAC1-PAC2 defines a specialized proteasome landscape in mature mammalian sperm

Cong, Y.; Zhou, X.; Shen, L.; Yuan, X.; Chen, C.; Wang, T.; Li, Z.; Peng, J.; Lang, X.; Ye, X.; Chen, T.; Chen, K.; Su, C.; Huang, Z.

doi:10.64898/2026.05.31.728944

bioRxiv · 10.64898/2026.05.31.728944

Repurposing of the assembly chaperone PAC1-PAC2 defines a specialized proteasome landscape in mature mammalian sperm

Cong, Y.·Zhou, X.·Shen, L.·Yuan, X.·Chen, C.·Wang, T.·Li, Z.·Peng, J.·Lang, X.·Ye, X.·Chen, T.·Chen, K.·Su, C.·Huang, Z.

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Abstract

Mature mammalian sperm rely on active proteasomes containing the testis-specific subunit 4s for capacitation, acrosomal exocytosis and fertilization, but how these proteasomes are structurally specialized remains unclear. We determine the endogenous proteasome landscape of mature bovine spermatozoa and testis using cryo-electron microscopy. We resolve five sperm proteasome assemblies: PA200-20S, free 20S, a previously unrecognized PA200-20S-PAC1/2 hybrid, and PAC1/2-20S complexes capped at one or both ends. Unexpectedly, the PAC1/2-bound complexes are mature and POMP-free, selectively incorporated for 4s, lack 2, and adopt a PAC1/2 orientation and gate configuration distinct from known assembly intermediates. Together with elevated proteolytic activity, these features indicate that PAC1/2 is repurposed from a transient assembly chaperone into a stable post-assembly regulator in mature sperm. 4s-containing proteasomes assemble through the canonical PAC1-PAC4/POMP pathway yet may follow {beta}2-{beta}3-{beta}1 incorporation ordering; functionally, 4s incorporation remodels the sCP surface and enhances peptidase activities, definining the spermatoproteasomes as a hyperactive 20S variant. PAC1/2-bound proteasomes are absent from testis, where PA200-20S and 26S proteasomes are present, while 26S proteasomes are nearly lost in mature sperm. Our findings define a sperm-specific, ATP-independent proteasome landscape, expand the functional repertoires of 4s and PAC1/2, and provide a structural framework for proteasome specialization in male fertility.

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Cong, Y., Zhou, X., Shen, L., Yuan, X., Chen, C., Wang, T., Li, Z., Peng, J., Lang, X., Ye, X., Chen, T., Chen, K., Su, C., Huang, Z.. 2026-06-02. Repurposing of the assembly chaperone PAC1-PAC2 defines a specialized proteasome landscape in mature mammalian sperm. https://doi.org/10.64898/2026.05.31.728944

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