Extensive cargo identification reveals distinct biological roles of the 12 importin pathways

Each sheet contains the result of SILAC-Tp with one of the 12 NTRs. The proteins that exhibited ${\displaystyle +NTR/Ctl=(L/H_{+NTR})/(L/H_{Ctl})}$ values at least once in the three replicates (Experiments 1–3) are listed with the ${\displaystyle {\mathrm{l}\mathrm{o}\mathrm{g}}_2[(\mathrm{L}/{\mathrm{H}}_{+\mathrm{N}\mathrm{T}\mathrm{R}})/(\mathrm{L}/{\mathrm{H}}_{\mathrm{C}\mathrm{t}\mathrm{l}})]}$ values. The second and third Z-scores and the ranks according to those scores are also presented. The 2nd-Z-15% and 3rd-Z-4% cargoes are indicated in cyan. The LC-MS/MS quantitation data for each replicate (Experiments 1–3) are also included. Light/Heavy, the median of quantified L/H values; Light/Heavy count, the number of quantified values; Light/Heavy variability, coefficient-of-variation for log-normal distributed data. ^aReport: The proteins listed by Chook and Süel (2011) are regarded as reported cargoes, and the references are provided in the Legend sheet. For Imp-β, only direct cargoes are listed. For Trn-2, the reported Trn-1 cargoes are listed. ^bDirect Binding: The results of the bead halo assays (Supplementary file 2) are summarized. ++ or +, positive; ± or –, negative. ^cGO Nucleus: Annotated with ‘nucleus’ in Gene Ontology (term type, cellular component). The rows can be sorted into preferable orders with Excel. To access the mass spectra, chromatograms, or raw data, see Supplementary file 12A. Statistics: For each experiment, the number of proteins assigned with ${\displaystyle {\mathrm{l}\mathrm{o}\mathrm{g}}_2[(\mathrm{L}/{\mathrm{H}}_{+\mathrm{N}\mathrm{T}\mathrm{R}})/(\mathrm{L}/{\mathrm{H}}_{\mathrm{C}\mathrm{t}\mathrm{l}})]}$ values (proteins assigned with Z-scores), the mean and standard deviation (S.D.) of ${\displaystyle {\mathrm{l}\mathrm{o}\mathrm{g}}_2[(\mathrm{L}/{\mathrm{H}}_{+\mathrm{N}\mathrm{T}\mathrm{R}})/(\mathrm{L}/{\mathrm{H}}_{\mathrm{C}\mathrm{t}\mathrm{l}})]}$ are listed. ${\displaystyle Z-score=(X-\mu )/\sigma }$ where ${\displaystyle X={\mathrm{l}\mathrm{o}\mathrm{g}}_2[(\mathrm{L}/{\mathrm{H}}_{+\mathrm{N}\mathrm{T}\mathrm{R}})/(\mathrm{L}/{\mathrm{H}}_{\mathrm{C}\mathrm{t}\mathrm{l}})]}$ of each protein, ${\displaystyle \mu }$ is the mean of X in one experiment, and ${\displaystyle \sigma }$ is the S.D. of ${\displaystyle X}$.

The direct binding of the candidate cargoes to the NTRs was analyzed by bead halo assay. From well-characterized proteins that have not been reported as cargoes, (i) proteins ranked high (within the top 15% in the 2nd-Z-rankings or 4% in the 3rd-Z-rankings), around presumptive cutoffs (within about top 15–25% in the 2nd-Z-rankings), or lower and (ii) highly ranked proteins that are suspected as indirect cargoes or false positives based on their well-known features, e.g., PMPCA, GALE, UAP1, NQO2, EEF1A2, RAB2A, RAB8A, S100A4, S100A6, S100A13, and S100P, were selected and analyzed. Proteins in (i) verify the cargo identification and cutoff setting, and proteins in (ii) serve for finding indirect cargoes and false positives. The negative rate of these bead halo assays should be higher than the true overall false positive rate of the SILAC-Tp, because proteins in (ii) were selected preferentially. GST or GST-NTR was attached to glutathione-Sepharose beads, mixed with an extract of E. coli expressing GFP or a GFP-fusion protein, and observed by fluorescence microscopy. Q69L-Ran, which inhibits the NTR–cargo functional binding, was added as appropriate. The contrast of the bead fluorescence between the GST and GST-NTR indicates the binding, and the inhibition of this binding by Q69L-Ran certifies the specificity of the binding; ++ or +, positive; ± or –, negative. Summary of the results (p2–5): The results are summarized in both 2nd- and 3rd-Z-rank order. The 2nd-Z-15% and 3rd-Z-4% cargoes are indicated by cyan, and positive binding (++ or +) is indicated by blue. Trn-1 (p6–8): The GFP-fusion proteins were divided into five groups (A–E) according to the expression levels. Because GFP binds weakly to Trn-1, the concentrations of GFP (control) and GFP-fusion proteins were equalized within each group, and the binding was observed in the same conditions. The images are comparable within a group. Trn-2 (p9): GFP weakly binds to Trn-2, and the concentrations of GFP and GFP-fusion proteins were equalized. The images are comparable. Proteins whose ranks differed substantially between the Trn-1 and Trn-2 Z-ranking were assayed. Imp-13 (p10–13): GFP does not bind to Imp-13, and GFP was added to the control mixture at the highest concentration. Three images (GST, GST-Imp-13, and GST-Imp-13 + Q69L-Ran) for each GFP-fusion protein were acquired under identical conditions, and the background intensities and dynamic ranges were equalized. Trn-SR (p14–16): GFP weakly binds to Trn-SR, and the procedures were similar to those used for Trn-1. The GFP-fusion proteins were divided into four groups (A–D), and the images are comparable within a group. Imp-α/β (p17–20): GFP does not bind to Imp-α or -β, and the procedures were similar to those used for Imp-13. GST-Imp-α₂ lacks the N-terminal Imp-β-binding domain. Western blotting (p21–22): The GFP-fusion proteins in the E. coli extracts were relatively quantified by Western blotting using an anti-GFP antibody (Roche). The extracts containing the amounts of protein (ng) indicated at the bottoms were loaded. The arrowheads indicate the expected full-length products. The GFP-moieties including those of the partial products were quantified by chemiluminescence. GFP was used as the standard. The Western blots were replicated more than three times. Accessions and sequences (p23–27): The cDNAs were cloned from a HeLa cDNA library by PCR. The accession numbers of the proteins are listed. If the sequence of a used protein is different from that in the database, the deleted, substituting, or inserted amino acids are indicated by the colors. The sequences that matched perfectly are not presented.

The 2nd-Z-15% cargoes of each NTR are listed by the gene names in the 2nd-Z-rank orders. The ranks by the third Z-scores are also shown. Cyan in the rank columns indicates the 2nd-Z-15% and 3rd-Z-4% cargoes. Colors in the gene name columns: magenta, reported cargoes; blue, cargoes bound directly to the NTR in the bead halo assays (Supplementary file 2); light blue, cargoes bound directly to Imp-α but not Imp-β; gray, proteins that did not bind to the NTRs; yellow, Imp-α; and green, reported export cargoes. For the 3rd-Z-4% cargoes, see Figure 3.

EICs of GLUD1 peptides in the SILAC-Tp with Trn-1: As a general problem of high-throughput LC-MS/MS quantitation, quantitative values of proteins with fewer quantified peptides deviate among the replicates. Referring to EICs of the quantified peptides is useful to avoid misidentification of cargoes. For example, the L/H ratios of a Trn-1 2nd-Z-15% cargo GLUD1 (P00367, ranked 110th and 342nd by the second and third Z-score, respectively) deviated largely in the three replicates of SILAC-Tp with Trn-1 (Supplementary file 1). Panels (A–H) show EICs of the indicated peptide (trypsin targets, K and R, are written in lower cases) in the three (three Ctl and there +Trn) experiments (some peptides were not identified in all the experiments). Magenta letters indicate the quantified peptides and L/H rations. In panel (A), the elution time of the peptide TAMkYNLGLDLr differs largely between the expriment-1 Ctl and experiment-2 +Trn-1, the peak shape of the experiment-2 +Trn-1 is irregular, and the L/H ratio of it is much higher than those of other peptides in +Trn-1 experiments (B and E). Thus, there is concern about misidentification. Because the L/H count of GLUD1 in the experiment-2 +Trn-1 is two (TAMkYNLGLDLr and NLNHVSYGr, A and B) and the L/H ratio of a protein is defined as the median, the L/H ratio of GLUD1 in the expriment-2 +Trn-1 is affected by the L/H ratio of TAMkYNLGLDLr. Exclusion of the L/H ratio of TAMkYNLGLDLr in the experiment-2 +Trn-1 lowers the Z-score rank of GLUD1 significantly. In panel (C), the chromatogram of the peptide HGGTIPIVPTAEFQDr in the experiment-1 Ctl has an irregular peak, and the L/H ratio of it is much higher than those of other peptides in the Ctl experiments (A–H). Thus, overlap with other peptide or other failures may be possible. However, the L/H count of GLUD1 in the experiment-1 Ctl is four (TAMkYNLGLDLr, HGGTIPIVPTAEFQDr, ALASLMTYk, and GASIVEDkLVEDLr) and the value of HGGTIPIVPTAEFQDr does not affect the median. (The L/H ratio of TAMkYNLGLDLr, whose EIC differ between the experiment-1 Ctl and expriment-2 +Trn in (A) as mentioned above, may affect the L/H ratio of GLUD1 in the experiment-1 Ctl, but we assumed that it is reliable.) As above, the L/H ratios of proteins with low L/H counts (Supplementary file 1) may be affected by LC-MS/MS artifacts, and misidentification can be avoided by referring to the EICs. All the EICs and MS spectra in this work can be accessed by downloading the mass spectrometry data and Proteome Discoverer software (see the Materials and methods and Supplementary file 12A).

(A) The numbers of the 2nd-Z-15% cargoes shared by two NTRs. For the 3rd-Z-4% cargoes, see Figure 4. (B) Redundancy of the 3rd-Z-4% cargoes. Many proteins are included in the 3rd-Z-4% cargoes of multiple NTRs. The ranks of these cargoes in the 3rd-Z-rankings for all 12 NTRs are presented. (C) Relationships between the NTRs and the characteristics of their 3rd-Z-4% cargo proteins. The 3rd-Z-4% cargoes are grouped according to their characteristics (functions or biological processes that the proteins act in), and their ranks are presented as in (B). To make our points clear, typical terms for the protein characteristics and typical proteins related to the terms have been selected with reference to Gene Ontology (GO) and UniProt. Thus, the terms in this sheet are slightly different from those in the databases, fewer proteins than annotated in the databases are grouped, and the list is redundant. For the complete linkages between the GO terms and the 3rd-Z-4% cargoes, see Supplementary file 7. (D) Redundancy of the 2nd-Z-15% cargoes. The ranks of the 2nd-Z-15% cargoes are presented as in (B). (E) Relationships between the NTRs and the characteristics of their 2rd-Z-15% cargo proteins. The 2nd-Z-15% cargoes are grouped, and their ranks are presented in a manner similar to that in (C). For the complete linkages between the GO terms and the 2nd-Z-15% cargoes, see Supplementary file 8.

(A) Extraction of the GO term enrichments of the 2nd-Z-15% cargoes. The 2nd-Z-15% cargoes were analyzed for GO term enrichment in (C). The terms that were significantly enriched (p<0.05, cyan) in the 2nd-Z-15% cargoes of four or fewer NTRs were selected, and terms that represent many similar terms are presented. With the p-values, the numbers (#) of cargoes annotated with each of the terms are presented. Total No. represents the number of proteins annotated with each term in the database. Related terms are bundled in the same color. For the 3rd-Z-4% cargoes, see Figures 5 and 6. The correspondences between each 2nd-Z-15% cargo and GO term are summarized in Supplementary file 10. All the GO terms annotated to the 2nd-Z-15% cargoes are listed in Supplementary file 8. (B) Full table of the GO term enrichments of the 3rd-Z-4% cargoes. The 3rd-Z-4% cargoes were analyzed for GO term enrichment. For all combinations of GO terms and NTRs, the p-values for the term enrichments in the 3rd-Z-4% cargoes and the numbers (#) of cargoes annotated with the terms are presented. The numbers following ‘# in’ are the total numbers of 3rd-Z-4% cargoes. Total No. represents the number of proteins annotated with each term in the database. Cyan, p<0.05. Figures 5 and 6 were extracted from this table. This table was derived from Supplementary file 7, and see Supplementary file 7 to retrieve the protein accessions. (C) Full table of the GO term enrichments of the 2nd-Z-15% cargoes. The 2nd-Z-15% cargoes were analyzed and are presented in a manner similar to that in (B). (A) was extracted from this table. This table was derived from Supplementary file 8, and see Supplementary file 8 to retrieve the protein accessions.

With respect to each NTR, the accessions of the 3rd-Z-4% cargoes annotated with each GO term are listed. Cyan, significant term enrichment (p<0.05) in the 3rd-Z-4% cargoes of the NTR. Total No. represents the number of proteins annotated with each term in the database. Supplementary files 6B and 9 were derived from this table. For the 2nd-Z-15% cargoes, see Supplementary file 8.

With respect to each NTR, the accessions of the 2nd-Z-15% cargoes annotated with each GO term are listed. Cyan, significant term enrichment (p<0.05) in the 2nd-Z-15% cargoes of the NTR. Total No. represents the number of proteins annotated with each term in the database. Supplementary files 6C and 10 were derived from this table. For the 3nd-Z-4% cargoes, see Supplementary file 7.

Each sheet shows the correspondences between the 3rd-Z-4% cargoes of one NTR and selected GO terms. A term annotation to a cargo is indicated by ‘1’ in the corresponding cell. Reported cargoes are indicated by magenta in the gene name cells, and the results of the bead halo assays (Supplementary file 2) are also indicated by colors in the gene name cells: blue, cargoes directly bound to the NTR; light blue, cargoes directly bound to Imp-α but not Imp-β; gray, proteins that did not bind to the NTR. GO terms that represent many similar terms were selected from the terms enriched significantly (p<0.05) for the 3rd-Z-4% cargoes of each NTR, and broadly defined terms were deselected. Magenta and orange in the term ID cells indicate terms that are significantly enriched for the cargoes of four or fewer NTRs, and of them magenta indicates the terms presented in Figures 5 and 6. Related GO terms are bundled in the same color, and different colors are used to distinguish the columns easily. The NTRs added in the transport reactions (white in the rank cells) were not analyzed. This table was derived from Supplementary file 7. For 2nd-Z-15% cargoes, see Supplementary file 10.

Each sheet shows the correspondences between the 2nd-Z-15% cargoes of one NTR and selected GO terms. A term annotation to a cargo is indicated by ‘1’ in the corresponding cell. Reported cargoes are indicated by magenta in the gene name cells, and the results of the bead halo assays (Supplementary file 2) are also indicated by colors in the gene name cells: blue, cargoes directly bound to the NTR; light blue, cargoes directly bond to Imp-α but not Imp-β; gray, proteins that did not bind to the NTR. GO terms that represent many similar terms were selected from the terms enriched significantly (p<0.05) for the 2nd-Z-15% cargoes of each NTR, and broadly defined terms were deselected. Magenta and orange in the term ID cells indicate terms that are significantly enriched for the cargoes of four or fewer NTRs, and of them magenta indicates the terms presented in Supplementary file 6A. Related GO terms are bundled in the same color, and different colors are used to distinguish the columns easily. The NTRs added in the transport reactions (white in the rank cells) were not analyzed. This table was derived from Supplementary file 8. For 3rd-Z-4% cargoes, see Supplementary file 9.

(A) Ranks of mRNA processing factors. All of the 2nd-Z-15% and 3rd-Z-4% cargoes that are annotated with mRNA processing in Gene Ontology (GO) are listed with the ranks in the 2nd- and 3rd-Z-rankings. The color scale is set by percentile rank as indicated. Figure 7 was extracted from this table. (B) The 3rd-Z-rankings of ribosomal proteins. The ranks of the ribosomal proteins in the 3rd-Z-rankings of the 12 NTRs are presented. The color scale is set by percentile rank as indicated. For the 2nd-Z-rankings, see Figure 8. (C) Extracts of the GO term enrichments of the transcription factors found in the 2nd-Z-15% cargoes. Seventeen to 36 proteins in the 2nd-Z-15% cargoes of each NTR are annotated with ‘transcription factor activity, sequence-specific DNA binding' or ’transcription factor activity, protein binding’ in GO. The factors were analyzed for GO term enrichment (term type, biological process, BP) in (D). Typical terms that represent similar terms were extracted from (D). The p-value for the term enrichment and the number (#) of factors annotated with the term are presented. Total No. represents the number of proteins annotated with each term in the database. Cyan, p<0.05. Related terms are bundled in the same color. (D) Full table of the GO term enrichments of the transcription factors found in the 2nd-Z-15% cargoes. The 2nd-Z-15% cargoes that are annotated with ‘transcription factor activity, sequence-specific DNA binding" or ’transcription factor activity, protein binding’ in GO were analyzed for GO term enrichment (term type, BP). The analyzed transcription factors are listed at the bottom. For all of the combinations of GO terms and NTRs, the p-value for the term enrichment and the number (#) of transcription factors annotated with the term are presented. The numbers following ‘# in’ are the total numbers of transcription factors in the 2nd-Z-15% cargoes. Total No. represents the number of proteins annotated with each term in the database. Cyan, p<0.05. (C) was extracted from this table. (E) SRSRSR motif in the 3rd-Z-4% cargoes. The 3rd-Z-4% cargoes that contain an ‘SRSRSR’ hexa-peptide sequence were counted.

(A) MS data files. The mass spectrometry proteomics data (.msf and .raw files) have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org/) with the dataset identifier PXD004655. The results of protein and peptide identification and quantitation are summarized in Supplementary file 1, and the .msf and .raw data files corresponding to each experiment in Supplementary file 1 are listed in this table. The quantitation results can be seen by opening .msf files by Proteome Discoverer software. To see spectra and chromatograms, .msf files and corresponding .raw files must be in the same local directory. A demo version of Proteome Discoverer can be downloaded at the Thermo Scientific omics software portal site (https://portal.thermo-brims.com/). (B) GFP-fusion proteins used for in vitro transport and antibodies.