Figure 6.

Higher-order assemblies of human CTLH E3

(A) Color-coded guide to yeast GID subunits and their human orthologs in the CTLH complex (top). Two colors indicate multiple protomers of a subunit. Cartoon colored as in the top, representing CTLH oval assembly where the SA module is the WDR26-YPEL5 dimer (bottom).

(B) 3.2-Å-resolution segmented map of CTLH SRS module (RANBP9-TWA1-ARMC8-hGid4) obtained by focused refinement of CTLH^SR4 (top) and its corresponding model (bottom).

(C) Cryo-EM maps of CTLH assemblies containing the Cat (RMND5A-MAEA), SRS (RANBP9-TWA1-ARMC8 alone or bound to hGid4), and/or supramolecular assembly (WDR26 with or without YPEL5) modules, as indicated. Subunits are colored according to the guide in (A). Top left: low-resolution map of WDR26-mediated SA of CTLH (RANBP9-TWA1-ARMC8-MAEA-RMND5A-WDR26). Right: 6.5-Å-resolution map of the human CTLH SRS module (RANBP9-TWA1-ARMC8-hGid4) subcomplex with an SA module comprising WDR26-YPEL5. Bottom panel: the yeast Gid2-Gid9 structure in the corresponding CTLH Cat module.

(D) 10.4-Å-resolution map of the human CTLH SRS module with MKLN1 as the SA module. The second copy of the SRS module in the subcomplex is transparent.

(E) Immunoblots of fractions from sucrose gradients of K562 cell lysates, probed with the indicated antibodies.

(F) Immunoblots probing for the core CTLH subunit (RANBP9) in fractions from sucrose gradients of lysates from parental K562 and WDR26^−/−/MKLN1^−/−, MKLN1^−/−, WDR26^−/−, and MAEA^−/− knockout cells. Black boxes delineate high- and low-molecular weight (MW) peak fractions.

(G) As in (F) but probed as indicated with anti-MKLN1 or -WDR26 antibodies. ^∗, WDR26 band.

See also Figures S6 and S7 and Table S1.