Architecture and mechanism of the human p24 cargo receptor

The p24 transmembrane emp24 domain family of cargo receptors is central to secretory trafficking, yet its molecular organization and regulatory principles have remained elusive. Here, we present cryo-EM structures of human p24 complex in three pH states, integrated with systematic functional analyses focused on glycosylphosphatidylinositol-anchored protein (GPI-AP) cargo. We identify a tightly interlocked p24α2-p24β1-p24δ1-p24γ2 heterotetramer as the minimal unit for GPI-AP engagement, resolving long-standing uncertainties regarding p24 stoichiometry and higher-order assembly. We reveal functional redundancy between p24α2 and p24α3, alongside selective incorporation of p24γ2 required for cargo binding. Systematic screening identifies two stable subassemblies: p24β1-p24δ1 and p24α2-p24β1-p24δ1, suggesting a stepwise assembly pathway. Comparative structural analysis across multiple pH states uncovers a conserved proton-sensing network centered on Coiled-coil layers 0 and +1 that drives coordinated transmembrane rearrangements, underlying pH-dependent cargo release. Our findings establish an integrated framework linking p24 architecture, isoform diversity, and pH regulation, offering a basis for understanding p24 trafficking complexity.