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.

Keywords: Importin; Mass Spectrometry (MS); Nuclear Pore; Nuclear Transport; Nucleocytoplasmic Transport; Nucleus; Protein-Protein Interactions; Proteomics; SILAC; Transportin.

FIGURE 1.

αβ/C values predict Imp-α/β cargoes. A, frequency distribution of L/H_Ctl ratios. Both L/H_Ctl and L/H_αβ ratios were calculated for 307 proteins, and distribution of the 307 L/H_Ctl ratios is illustrated. B, protein ranking by a reliable αβ/C value. Proteins quantified reliably by MS (88 proteins with 0.3 < L/H_Ctl < 1.1 and 0.03 < L/H_αβ) were ranked in descending αβ/C value order (Table 1). Horizontal, protein rank; vertical, αβ/C value. IMB1 (Imp-β) ranked at the top is not shown. The results of the bead halo assays are indicated by the following colors: magenta, bound to Imp-α or Imp-β directly; violet, bound to Imp-α via another cargo protein; blue, unclear; cyan, not bound to Imp-α or Imp-β; and black, not assayed.

FIGURE 2.

Interaction of candidate cargoes with Imp-α or Imp-β. Cargo-carrier interactions were analyzed by a bead halo assay. Bacterial extract containing a GFP fusion protein was mixed with GSH-Sepharose beads and one of GST, GST-Imp-αΔN, GST-Imp-β, Imp-α/GST-Imp-β, or Imp-α/GST-Imp-β/Q69L-Ran. The interactions were observed by fluorescence microscopy. The extracts were normalized for the GFP moiety and total protein concentration following quantitative Western blotting (supplemental Fig. S1). Q69L-Ran inhibits specific interactions between Imp-β and the cargo or Imp-α. The images within each panel are comparable. Protein numbers are identical to those in Table 1. A, cargo proteins that bound very strongly to Imp-α. The exposure time was shorter than that for panel B. B, images taken using standard conditions. C and D, enhanced images for proteins 6, 10, 18, and 32. These images show faint but detectable signals.

FIGURE 3.

Cargo complexes. Candidate cargoes that did not bind directly to Imp-α or Imp-β (Fig. 2) were examined for indirect binding via other cargoes by a bead halo assay. A, components of the MCM2–7 complex. GFP-MCM6 was assayed for binding to Imp-α or Imp-β in the presence or absence of intact MCM2, which binds to Imp-α (Fig. 2). B, components of the spliceosome C or mRNP granule complex. GFP-ROA2 was assayed in the presence or absence of intact HNRPU or HNRPK, which bind to Imp-α (Fig. 2). Assays were conducted as described in the legend to Fig. 2, but the images are enhanced. Images within each panel are comparable.

FIGURE 4.

cNLS sequences in the identified proteins. NLS scores, which were defined by the cNLS mapper program, were calculated and are shown for proteins listed in Table 1. For proteins with two or more predicted cNLS sequences, the highest NLS score was used. Vertical, NLS score; horizontal, protein rank in Table 1. The results of the bead halo assays are indicated by the following colors: filled magenta, bound to Imp-α; open magenta, bound to Imp-β; violet, bound to Imp-α indirectly; blue, unclear because of degradation; cyan, not bound to Imp-α or Imp-β; and gray, not assayed. Roughly, exclusive nuclear or cytoplasmic localization is predicted by a score of >8 or <3 (dashed lines), respectively.

FIGURE 5.

Identified Imp-α/β and Trn cargoes. A, αβ/C and T/C values. Log₂(αβ/C) is plotted against log₂(T/C) for 77 proteins that were quantified based on highly reliable quality control values (0.03 < L/H_Ctl < 1.1, 0.03 < L/H_Trn, and 0.03 < L/H_αβ). The following colors indicate the results of the bead halo assay: orange, bound to Trn but not to Imp-α or Imp-β; magenta, bound to Imp-α or Imp-β but not to Trn; green, bound to both; black, not assayed or not bound to either. Proteins with prominent values are labeled. B and C, bi-pathway cargoes. Green letters, bi-pathway cargo; magenta letters, Imp-β cargo; orange arrow, protein-protein interaction suggestive of the Trn pathway; and magenta arrow, suggestive of the Imp-β pathway. NLS types engaging in the interactions are denoted.