title: Identification of cargo proteins specific for importin-β with importin-α applying a stable isotope labeling by amino acids in cell culture (SILAC)-based in vitro transport system.
abstract: The human importin (Imp)-β family consists of 21 nucleocytoplasmic transport carrier proteins, which transport thousands of proteins (cargoes) across the nuclear envelope through nuclear pores in specific directions. To understand the nucleocytoplasmic transport in a physiological context, the specificity of cargoes for their cognate carriers should be determined; however, only a limited number of nuclear proteins have been linked to specific carriers. To address this biological question, we recently developed a novel method to identify carrier-specific cargoes. This method includes the following three steps: (i) the cells are labeled by stable isotope labeling by amino acids in cell culture (SILAC); (ii) the labeled cells are permeabilized, and proteins in the unlabeled cell extracts are transported into the nuclei of the permeabilized cells by a particular carrier; and (iii) the proteins in the nuclei are quantitatively identified by LC-MS/MS. The effectiveness of this method was demonstrated by the identification of transportin (Trn)-specific cargoes. Here, we applied this method to identify cargo proteins specific for Imp-β, which is a predominant carrier that exclusively utilizes Imp-α as an adapter for cargo binding. We identified candidate cargoes, which included previously reported and potentially novel Imp-β cargoes. In in vitro binding assays, most of the candidate cargoes bound to Imp-β in one of three binding modes: directly, via Imp-α, or via other cargoes. Thus, our method is effective for identifying a variety of Imp-β cargoes. The identified Imp-β and Trn cargoes were compared, ensuring the carrier specificity of the method and illustrating the complexity of these transport pathways.
authors.affiliation: Cellular Dynamics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. makimura@riken.jp
authors.forename: Makoto
authors.initials: M
authors.lastname: Kimura
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Okumura
authors.forename: Shingo
authors.initials: S
authors.lastname: Kose
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Takao
authors.forename: Naoko
authors.initials: N
authors.lastname: Imamoto
mesh_terms: D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D021381:Protein Transport / Q000502:physiology; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism*
chemical_list: D028884:Karyopherins; D028901:alpha Karyopherins; D028961:beta Karyopherins
keywords: Importin; Mass Spectrometry (MS); Nuclear Pore; Nuclear Transport; Nucleocytoplasmic Transport; Nucleus; Protein-Protein Interactions; Proteomics; SILAC; Transportin