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-␤ 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. Most genetic processes, including chromosome replication and transcription, occur in the nucleus, and thus the selection of proteins that enter the nucleus and act there is crucial for cellular processes (1). During interphase, all nuclear proteins cross the nuclear envelope via nuclear pores, channels that constitute a selective permeability barrier for macromolecules. Only a fraction of proteins traverse these channels by means of free diffusion; most nuclear proteins are imported into or exported out of the nucleus with the assistance of importin-␤ (Imp-␤) 1 family proteins (Imp-␤s) (2, 3). Imp-␤s interact with the nuclear pore complex in a way that allows them to travel in and out of the nucleus. To drive cargo From the ‡Cellular Dynamics Laboratory, Advanced Science Institute, RIKEN, 2-1 Hirosawa,

Landscape of nuclear transport receptor cargo specificity

“…Systematic approaches to measure the cargo spectrum in an NTR‐centric manner have been reported, but were often limited to a single NTR (XPO1; Thakar et al , 2013; Kırlı et al , 2015), or conducted ex vivo . The import rate of SILAC‐labeled cellular extracts has been measured using mass spectrometry in the presence and absence of various importin βs to chart their cargo spectrum (Kimura et al , 2013a, 2014, 2017). This work was performed in permeabilized cells in which the cytoplasmic membrane has been punctured and the cytosolic content washed out.…”

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*

“…Buffers, the ATP regeneration system, antibodies, cytosolic, and nuclear extract preparations, and the evaluation of the transport system were described previously (12).…”

“…Sequences are described for proteins whose cDNA sequences were not present in the database. GFP fusion proteins were expressed in Escherichia coli, and the extracts were prepared as described previously (12). The GFP moiety in the E. coli extracts was quantified three times by Western blotting (supplemental Fig.…”

“…One advantage of this method is that cargo proteins accumulate in the nuclei regardless of whether the cargo-carrier interaction is short-lived or dissociates during pulldown experiments. For Trn, the SILAC-Tp method identified a set of cargoes that partly overlapped, but differed considerably from those identified by other binding-based methods, indicating that our method can complement conventional methods (12).…”

“…To date, only a limited number of cargoes have been linked to specific carriers, and some carriers have no validated cargo proteins. To address this issue, we recently developed a novel SILAC-based (11) in vitro transport (SILAC-Tp) method to identify carrier-specific cargoes, and this method identified Trn-specific cargoes (12). The method was validated using bead halo assays (13), which detect the physical interactions between the identified cargoes and Trn.…”