Oral Presentation The 45th Lorne Conference on Protein Structure and Function 2020

Systematic knockout of Commander subunits reveals novel assembly modules and specific roles for COMMD subunits in retriever-dependent endosomal sorting (#53)

Joanna Sacharz 1 , Calum McConville 1 , Nikeisha Caruana 1 , Michael D Healy 2 , Rajesh Ghai 1 2 , Brett M Collins 2 , David A Stroud 1
  1. Department of Biochemistry and Molecular Biology and The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia
  2. Division of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia

Following endocytosis into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are recycled back to the cell surface. While the importance and roles of the retromer complex are well-established in this process, the recently identified retriever complex1 has emerged as a critical regulator of endosomal sorting. Although retriever shares striking similarities to retromer, its cargo-specificity appears to be distinct. Retriever associates with ten members of the Copper Metabolism MURR1 (Mouse U2af1-rs1 region 1) Domain-containing (COMMD) family and two Coiled Coil Domain Containing (CCDC) family members to form a large multi-subunit assembly that when associated with retriever is known as Commander. While the structural mechanism of COMMD-COMMD association is now understood2, little else is known about the assembly, interdependency or the stoichiometries of subunits within the Commander complex. We employed systematic CRISPR/Cas9-mediated deletion of all individual Commander subunits combined with blue-native (BN)-PAGE and quantitative proteomic analysis to define structural modules and understand complex biogenesis, as we have done previously with other large multi-subunit assemblies3. We found that Commander assembles into a stable 700 kDa complex, containing all ten COMMD and both CCDC proteins. We identify and define several different sub-assemblies which may act as precursors for the assembly of the mature complex. Loss of selected COMMD proteins does not lead to complete breakdown of the complex, but rather alters the cargo specificity of Commander, suggesting specific roles for individual COMMD proteins in endosomal trafficking.

 

1McNally K.E. et al., Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling (2017) Nat. Cell Biol., 19:1214-1225.

2Healy M.D. et al., Structural insights into the architecture and membrane interactions of the conserved COMMD proteins (2018) eLife, 7.

3Stroud D.A. et al., Accessory subunits are integral for assembly and function of human mitochondrial complex I (2016) Nature, 538:123-126