A USP28-TM9SF3-Golgiphagy axis maintains glycosylation homeostasis and promotes breast cancer progression.

Selective autophagy of the Golgi apparatus (Golgiphagy) is essential for Golgi quality control, yet its regulation and physiological functions remain poorly understood. Here, we identify the deubiquitinase USP28 as an upstream regulator of the Golgiphagy receptor TM9SF3. USP28 removes degradative K48-linked ubiquitin chains from TM9SF3, preventing its proteasomal degradation and sustaining Golgiphagy under nutrient and Golgi stresses. CRM1-dependent nuclear export enables the stress-induced translocation of USP28 to the Golgi, where it stabilizes TM9SF3, promoting Golgi fragmentation and autophagic clearance. Loss of USP28 or TM9SF3 impairs Golgiphagy, disrupts glycosylation fidelity, and inhibits breast cancer cell proliferation. Proteomic and functional analyses identify the glycosyltransferase MGAT5 as a selective TM9SF3-dependent Golgiphagy cargo, revealing how this pathway maintains glycosylation homeostasis. In syngeneic tumor models, USP28 knockout or pharmacological inhibition reduces TM9SF3 abundance, compromises Golgiphagy, perturbs glycosylation, and markedly suppresses tumor growth-effects restored by wild-type but not LC3-binding-deficient TM9SF3. Together, these findings reveal a USP28-TM9SF3-Golgiphagy axis that couples ubiquitin signaling to Golgi glycosylation control and represents a therapeutically targetable pathway for breast cancer.