Fig. 2. The N-terminal “belt” sequence unique to VPS35L is key to Retriever assembly.

(A) Cryo-EM density of the “belt” sequence interacting with VPS35L and VPS29. (B) Alignment of the “belt” sequences from representative species from animal to amoeba and plants. Residues shown in (A) are marked with arrowheads. (C-D) Key interactions between the “belt” sequence (represented in cartoon, with carbon in green, oxygen in red, and nitrogen in blue) and its binding surface on VPS35L (C) and VPS29 (D). The binding surface is colored based on conservation score calculated by Consurf⁷¹, with color to white gradients representing the most (ConSurf score = 9) to the least conserved residues (ConSurf score = 1). Contacting residues are shown as sticks. Yellow dashed lines indicate polar interactions. (E-F) Immunoprecipitation of VPS35L NT-deletion mutants expressed in HEK293T cells. Interactions with indicated components of Retriever and CCC were assessed by immunoblotting.