Structural Mechanisms in Retrograde Protein Traffic to the Golgi

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Structural Mechanisms in Retrograde Protein Traffic to the Golgi The Golgi apparatus is the eukaryotic cells central sorting depot. Retrograde traffic of lipids, Golgi-resident sorting receptors, SNAREs, and other proteins is required to maintain Golgi homeostasis and enable cyclical sorting pathways. One of the best characterized retrograde sorting pathways, conserved from yeast to humans, is the sorting of Vps10 in yeast and the mannose 6-phosphate receptors in humans by the retromer complex. Retromer consists of a cargo recognition complex (Vps26, Vps29, and Vps35), and a dimeric sorting nexin (SNX) complex that targets and tubulates membranes. Retromer is required for transcytosis of the polymeric Ig receptor and for Wnt gradient formation, among other processes. Despite its importance in multiple pathways, the molecular function of retromer remains somewhat obscure. To address this gap in knowledge, we seek to 1) determine the structures of retromer subunits by x-ray crystallography; 2) determine how the subunits interact with each other to form the intact complex using crystallography, scanning mutagenesis, hydrodynamics, small angle x-ray scattering, modeling, and electron microscopy; and 3) determine how retromer interacts with membranes and cargo using binding and structural studies. The retromer complex is required for the sorting of acid hydrolases to lysosomes, transcytosis of the polymeric Ig receptor, Wnt gradient formation, iron transporter recycling, and processing of the amyloid precursor protein. Human retromer consists of two smaller complexes, the cargo recognition Vps26:Vps29:Vps35 heterotrimer, and a membrane-targeting heterodimer or homodimer of SNX1 and/or SNX2. The retromer cargo recognition complex consists of the 38-kDa Vps26, 20-kDa Vps29, and 92-kDa Vps35 subunits. Studies under this project in 2006 showed that Vps26 is a structural cousin of the arrestins, a family of trafficking proteins that directly bind to cell surface receptors and direct their internalization. Juan Bonifacinos laboratory, working in collaboration with our group, showed that Vps26 binds to Vps35 through a conserved loop in the C-terminal lobe, and found that this loop is required for the correct localization of Vps26 in vivo. Vps29 was shown by two other labs to have a metallophosphoesterase fold that can bind two metal ions. Compared to functional metallophosphoesterases, a key His that serves as a catalytic base in the metallophosphoesterase active site is replaced by Phe 63. Thus Vps29 is completely inactive with respect to generic phosphatase substrates. However, metal-dependent activity in vitro against a phosphorylated peptide from a major retromer cargo, the cation-independent mannose 6-phosphate receptor (CI-MPR), has been reported. Despite its centrality to multiple trafficking pathways, the precise function of retromer has been enigmatic. Various proposals have emphasized potential roles as a coat, adaptor, or cargo protein phosphatase. The main accomplishment over the past FY was to determine the crystal structure of the SNX1 BAR domain homodimer. While SNX1 homodimers are not thought to function in retrograde sorting in cells, this structure provides and improved basis for generating models of the heterodimers and for understanding the preferential formation of SNX1/2/5/6 heterodimers.