Volume 14, Issue 4, Pages 661-671 (April 2006) The Endosome-Associated Protein Hrs Is Hexameric and Controls Cargo Sorting as a “Master Molecule”  Lee Pullan, Srinivas Mullapudi, Zhong Huang, Philip R. Baldwin, Christopher Chin, Wei Sun, Susan Tsujimoto, Steven J. Kolodziej, James K. Stoops, J. Ching Lee, M. Neal Waxham, Andrew J. Bean, Pawel A. Penczek  Structure  Volume 14, Issue 4, Pages 661-671 (April 2006) DOI: 10.1016/j.str.2006.01.012 Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 1 Purification of Oligomeric Hrs (A) Plot of the gel filtration of the cell lysate yielding oligomeric Hrs. The arrow indicates the 540 kDa hexameric Hrs peak. Molecular weight standards (dashed line), peaks A, B, C, D, and E, are thyroglobulin, 670 kDa; γ globulin, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa; and vitamin B12, 1.35 kDa, respectively. (B) The 90 kDa Hrs monomer, contained in the 540 kDa peak, is indicated by the arrow on the Coomassie-stained SDS gel (lane 1). The Western blot shows the reactivity of Hrs with an anti-Hrs monoclonal antibody (lane 2). The discrepancy of the location of the Hrs band in the gels is due to the presence of the hexa-his tag in the protein. (C) An AUC plot shows that the sample of Hrs is homogenous. Structure 2006 14, 661-671DOI: (10.1016/j.str.2006.01.012) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 2 Oligomeric Hrs Binds to SNAP-25 and Endosomal Membranes and Inhibits Endosomal Fusion (A) Purified Hrs was incubated with SNAP-25 (lane 1) or control (GST, lane 2) and washed, and proteins bound to sepharose were separated by SDS-PAGE. The resulting blots were probed with anti-Hrs antisera. (B) Oligomeric Hrs binds to early endosomal membranes. Early endosomes were purified and incubated with increasing concentrations of His-tagged oligomeric Hrs (lanes 1–5; 45, 90, 180, 360, 540 nM). His-Hrs was visualized in the pellet fraction by using anti-His antibodies. Oligomeric Hrs is not found in the pellet in the absence of membranes (not shown). (C) Hrs inhibits early endosome homotypic fusion in a dose-dependent and saturable manner (diamond). The concentrations required to inhibit fusion are similar to those previously reported (Sun et al., 2003). The error bars indicate the standard error of the mean for individual points. Structure 2006 14, 661-671DOI: (10.1016/j.str.2006.01.012) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 3 A Cryo-Micrograph of Native Hrs Two distinct shapes are: circular end views (Area 1) and rectangular side views (Area 2). The scale bar represents 50 nm. Structure 2006 14, 661-671DOI: (10.1016/j.str.2006.01.012) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 4 2D Class Averages and Eigenimages of Hexameric Hrs Particles (A) Side views. The short axis through the central core is indicated by the arrows in Average 1, and the long axis is indicated by the black arrows in Average 2. Averages 1, 2, and 3 have mirror symmetry about the short axis. (B) End views. The white arrows point to the locations of three mirror symmetry axes in Average 1. The scale bar represents 100 nm. (C) The first three eigenimages of the principal component analysis of the side views. Structure 2006 14, 661-671DOI: (10.1016/j.str.2006.01.012) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 5 3D Cryo-EM Structure of Hexameric Hrs (A) Side view of Hrs with holes in the central core and the oligomerization of the monomers within the antiparallel pair via the coiled-coil domains. The scale bar represents 50 nm. (B) End view of Hrs indicating the electron density for the FYVE and VHS domains, and the carboxy-terminal domain. Structure 2006 14, 661-671DOI: (10.1016/j.str.2006.01.012) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 6 The Known Functional Domains of Hrs (A) The Hrs primary protein sequence indicating the known functional domains. (B) Predicted fit of the domains of the Hrs monomer into the EM density map. The color scheme: VHS domain, red; FYVE domain, cyan; UIM domain, purple; the coiled-coil domains, green. The region encompassing amino acids (216–449) is reactive to antibody (star and arrow pointers). Structure 2006 14, 661-671DOI: (10.1016/j.str.2006.01.012) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 7 Fit of the Crystal Structure of the VHS and FYVE Domains into the EM Map (A) Cryo-EM map of Hrs with the VHS (red) and FYVE (yellow) domains shown as electron density maps. (B) Zoomed-in region of the VHS and FYVE domains shown as ribbon structure representations. Structure 2006 14, 661-671DOI: (10.1016/j.str.2006.01.012) Copyright © 2006 Elsevier Ltd Terms and Conditions