Reviewer #2 (Public review): A novel RAB5 binding site in human VPS34-CII that is likely the primordial site in eukaryotic evolution

RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII, by binding to a helical insertion in the C2 domain of VPS34 on the BECLIN1/UVRAG-containing adaptor arm of the complex. The autophagy complex, VPS34 complex I (VPS34-CI), features a unique ATG14L subunit in place of the VPS34-CII UVRAG subunit, and we found that this distorts the adaptor arm to alter the VPS34 RAB-GTPase binding pocket so that it preferentially binds RAB1-GTP. Surprisingly, our higher-resolution single-particle cryo-EM structure of VPS34-CII showed a second RAB5-GTP binding site on the VPS15 solenoid region. This site (VPS15-RAB5-site) appears to be the primordial RAB5-binding region. A mutant in the helical insertion of the C2 domain of human VPS34 that mimics the Saccharomyces cerevisiae sequence abolishes RAB5 binding to VPS34. Mutation of the VPS15-RAB5-site ortholog in S. cerevisiae VPS15 resulted in defective CPY sorting, loss of colocalization with the RAB5 ortholog Vps21, and loss of binding to Vps21 in vitro. Evolutionary expansion from one to two RAB5-orthologue binding sites may have increased membrane binding and VPS34-CII activity to adapt to more complex endocytic systems.