Cryo-EM structure of the WDR11-FAM91A1-C17orf75 complex involved in retrograde trafficking.

Retrograde trafficking from endosomes to trans-Golgi network is essential for cellular homeostasis. While the WDR11-FAM91A1-C17orf75 (WFC) complex facilitates this process, its structural organization and the role of C17orf75 remain unclear. Here, we present cryo-EM structures of human WFC complex in monomeric and dimeric forms at 3.34 Å and 3.07 Å resolution, respectively. The WFC complex forms a dimer through the α-solenoid domains of WDR11. FAM91A1 serves as a central scaffold that interacts directly with both WDR11 and C17orf75, whereas WDR11 and C17orf75 have no direct contact. C17orf75 features an N-terminal longin-like domain and a C-terminal DENN-like domain and contains a positively charged groove that may cooperate with WDR11 in recognizing acidic-cluster-containing cargo proteins. Functional analyses demonstrate that C17orf75 is indispensable for the proper intracellular localization of the acidic-cluster-containing cargo proteins CI-MPR, KIAA0319L, and VAMP4. These findings establish C17orf75 as an integral component of the WFC complex in endosome-to-TGN trafficking.