title: Integrative structure-function mapping of the nucleoporin Nup133 suggests a conserved mechanism for membrane anchoring of the nuclear pore complex.
abstract: The nuclear pore complex (NPC) is the sole passageway for the transport of macromolecules across the nuclear envelope. Nup133, a major component in the essential Y-shaped Nup84 complex, is a large scaffold protein of the NPC's outer ring structure. Here, we describe an integrative modeling approach that produces atomic models for multiple states of Saccharomyces cerevisiae (Sc) Nup133, based on the crystal structures of the sequence segments and their homologs, including the related Vanderwaltozyma polyspora (Vp) Nup133 residues 55 to 502 (VpNup133(55-502)) determined in this study, small angle X-ray scattering profiles for 18 constructs of ScNup133 and one construct of VpNup133, and 23 negative-stain electron microscopy class averages of ScNup133(2-1157). Using our integrative approach, we then computed a multi-state structural model of the full-length ScNup133 and validated it with mutational studies and 45 chemical cross-links determined via mass spectrometry. Finally, the model of ScNup133 allowed us to annotate a potential ArfGAP1 lipid packing sensor (ALPS) motif in Sc and VpNup133 and discuss its potential significance in the context of the whole NPC; we suggest that ALPS motifs are scattered throughout the NPC's scaffold in all eukaryotes and play a major role in the assembly and membrane anchoring of the NPC in the nuclear envelope. Our results are consistent with a common evolutionary origin of Nup133 with membrane coating complexes (the protocoatomer hypothesis); the presence of the ALPS motifs in coatomer-like nucleoporins suggests an ancestral mechanism for membrane recognition present in early membrane coating complexes.
authors.affiliation: From the ‡Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, Byers Hall, 1700 4th Street, Suite 503B, University of California San Francisco, San Francisco, California 94158;
authors.forename: Seung Joong
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: ¶Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065;
authors.forename: Javier
authors.initials: J
authors.lastname: Fernandez-Martinez
authors.affiliation: ‖Department of Biochemistry, Ullmann Building, Room 409, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461;
authors.forename: Parthasarathy
authors.initials: P
authors.lastname: Sampathkumar
authors.affiliation: **Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, MS 69, Menlo Park, California 94025;
authors.forename: Anne
authors.initials: A
authors.lastname: Martel
authors.affiliation: **Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, MS 69, Menlo Park, California 94025;
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Matsui
authors.affiliation: **Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, MS 69, Menlo Park, California 94025;
authors.forename: Hiro
authors.initials: H
authors.lastname: Tsuruta
authors.affiliation: **Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, MS 69, Menlo Park, California 94025;
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Weiss
authors.affiliation: ‡‡Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065;
authors.forename: Yi
authors.initials: Y
authors.lastname: Shi
authors.affiliation: §§Laboratorio de Genetica Molecular de Aspergillus, Departamento de Biología Celular y Molecular, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040, Madrid, Spain;
authors.forename: Ane
authors.initials: A
authors.lastname: Markina-Inarrairaegui
authors.affiliation: ‖Department of Biochemistry, Ullmann Building, Room 409, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461;
authors.forename: Jeffery B
authors.initials: JB
authors.lastname: Bonanno
authors.affiliation: ¶¶Discovery Chemistry Research and Technologies (DCR&T), Eli Lilly and Company, Lilly Biotechnology Center, 10300 Campus Point Drive, Suite 200, San Diego, California 92121;
authors.forename: J Michael
authors.initials: JM
authors.lastname: Sauder
authors.affiliation: ‖‖Center for Integrative Proteomics Research, Department of Chemistry and Chemical Biology, Rutgers, the State University of New Jersey, 174 Frelinghuysen Road, Piscataway, New Jersey 08854.
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Burley
authors.affiliation: ‡‡Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065;
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.affiliation: ‖Department of Biochemistry, Ullmann Building, Room 409, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461; steve.almo@einstein.yu.edu rout@rockefeller.edu sali@salilab.org.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.affiliation: ¶Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065; steve.almo@einstein.yu.edu rout@rockefeller.edu sali@salilab.org.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
authors.affiliation: From the ‡Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, Byers Hall, 1700 4th Street, Suite 503B, University of California San Francisco, San Francisco, California 94158; steve.almo@einstein.yu.edu rout@rockefeller.edu sali@salilab.org.
authors.forename: Andrej
authors.initials: A
authors.lastname: Sali
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D007716:Kluyveromyces / Q000201:enzymology*; D008958:Models, Molecular; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D017386:Sequence Homology, Amino Acid
chemical_list: C091271:NUP133 protein, S cerevisiae; C097894:NUP84 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D029701:Saccharomyces cerevisiae Proteins