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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.
Highlights
From the ‡Cellular Dynamics Laboratory, Advanced Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; ¶Laboratory of Homeostatic Integration, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan; ʈComputational Biology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Kotoku, Tokyo 135-0064, Japan; **Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo 102-8472, Japan; ‡‡Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Import carriers bind to their cargoes in the cytoplasm and travel to the nucleus, where they release their cargoes upon binding to RanGTP
Transport-based Cargo Identification System—Screening for candidate cargo proteins relies on the relative quantification of stable isotope-labeled “heavy” and unlabeled “light” peptides by means of MS, together with an in vitro reconstituted transport system (Fig. 1)
Summary
The buffers, ATP regeneration system, proteins, antibodies, cytosolic and nuclear extracts, evaluation of the transport system, and identification of Ran-regulated Trn-interacting proteins via a pulldown method are described in the supplemental data. The resulting permeabilized cells were pretreated with TB containing 4 M RanGDP and an ATP regeneration system for 20 min at 30 °C to remove residual Imp-s before the transport reaction. After rinsing with TB twice, the transport reaction was carried out through incubation of the cells in 250 l of transport mixture (50% Imp-depleted cytosolic extract, 10% Imp- and RCC1-depleted nuclear extract, 1 M p10/NTF2, and ATP regeneration system in TB) with (ϩTrn) or without (control) 0.3 M Trn for 20 min at 30 °C. After the mixtures had been incubated for 20 to 30 min, they were observed via fluorescence microscopy
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