Rheb ubiquitination drives LC3 lipidation via non-canonical autophagy to promote porcine sperm acrosome reaction.

The coordinated interaction between ubiquitination and autophagy is essential for maintaining cellular homeostasis; however, their precise roles in regulating the acrosome reaction (AR) remain unclear. To determine whether Rheb ubiquitination modulates LC3 lipidation through the mTORC1/ULK1-Beclin 1 signaling axis during sperm capacitation and thereby influences the AR, we conducted a series of molecular analyses in porcine sperm samples. These included quantitative ubiquitin proteomics, co-immunoprecipitation (CO-IP), Western blot (WB), and immunofluorescence. Our results revealed that capacitation markedly increased Rheb ubiquitination (P < 0.01), accompanied by a switch from degradative K48-linked to non-degradative K63-linked polyubiquitin chains. Upregulation of Rheb ubiquitination suppressed mTORC1 activity, thereby activating the ULK1-Beclin 1 signaling cascade and promoting LC3 lipidation (conversion to LC3-II). Lipidated LC3 accumulated at the acrosomal membrane and showed enhanced interaction with the vesicle-associated protein VAMP3 during AR (P < 0.01). Inhibition of LC3 lipidation reduced AR incidence (P < 0.05). These findings indicate that Rheb ubiquitination facilitates AR via a non-canonical autophagy pathway, likely through LC3 acrosomal localization and its interaction with VAMP3, offering new mechanistic insights into sperm function and male infertility.