title: Identification of cargo proteins specific for the nucleocytoplasmic transport carrier transportin by combination of an in vitro transport system and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics.
abstract: The human importin-β family consists of 21 nucleocytoplasmic transport carrier proteins that carry proteins and RNAs across the nuclear envelope through nuclear pores in specific directions. These transport carriers are responsible for the nucleocytoplasmic transport of thousands of proteins, but the cargo allocation of each carrier, which is necessary information if one wishes to understand the physiological context of transport, is poorly characterized. To address this issue, we developed a high-throughput method to identify the cargoes of transport carriers by applying stable isotope labeling by amino acids in cell culture to construct an in vitro transport system. Our method can be outlined in three steps. (1) Cells are cultured in a medium containing a stable isotope. (2) The cell membranes of the labeled cells are permeabilized, and proteins extracted from unlabeled cells are transported into the nuclei of the permeabilized cells. In this step, the reaction system is first depleted of all importin-β family carriers and then supplemented with a particular importin-β family carrier of interest. (3) Proteins in the nuclei are extracted and analyzed quantitatively via LC-MS/MS. As an important test case, we used this method to identify cargo proteins of transportin, a representative member of the importin-β family. As expected, the identified candidate cargo proteins included previously reported transportin cargoes as well as new potential cargoes, which we corroborated via in vitro binding assays. The identified cargoes are predominately RNA-interacting proteins, affirming that cargoes allotted to the same carrier share functional characteristics. Finally, we found that the transportin cargoes possessed at least two classes of signal sequences: the well characterized PY-nuclear localization signals specific for transportin, and Lys/Arg-rich segments capable of binding to both transportin and importin-β. Thus, our method will be useful for linking a carrier to features shared among its cargoes and to specific nuclear localization signals.
authors.affiliation: Cellular Dynamics Laboratory, Advanced Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. makimura@riken.jp
authors.forename: Makoto
authors.initials: M
authors.lastname: Kimura
authors.forename: Shingo
authors.initials: S
authors.lastname: Kose
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Okumura
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Imai
authors.forename: Maiko
authors.initials: M
authors.lastname: Furuta
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sakiyama
authors.forename: Kentaro
authors.initials: K
authors.lastname: Tomii
authors.forename: Paul
authors.initials: P
authors.lastname: Horton
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Takao
authors.forename: Naoko
authors.initials: N
authors.lastname: Imamoto
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000596:Amino Acids; D002462:Cell Membrane; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling; D009685:Nuclear Envelope / Q000378:metabolism; D019913:Nuclear Localization Signals / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D028961:beta Karyopherins / Q000032:analysis* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D019913:Nuclear Localization Signals; C108121:TNPO1 protein, human; D028961:beta Karyopherins