October 19, 2015;
The Nuclear Proteome of a Vertebrate.
The composition of the nucleoplasm determines the behavior of key processes such as transcription, yet there is still no reliable and quantitative resource of nuclear proteins. Furthermore, it is still unclear how the distinct nuclear and cytoplasmic compositions are maintained. To describe the nuclear proteome quantitatively, we isolated the large nuclei of frog oocytes via microdissection and measured the nucleocytoplasmic partitioning of ∼9,000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus
; only ∼17% partition equally. A protein''s native size in a complex, but not polypeptide molecular weight, is predictive of localization: partitioned proteins exhibit native sizes larger than ∼100 kDa, whereas natively smaller proteins are equidistributed. To evaluate the role of nuclear export in maintaining localization, we inhibited Exportin 1
. This resulted in the expected re-localization of proteins toward the nucleus
, but only 3% of the proteome was affected. Thus, complex assembly and passive retention, rather than continuous active transport, is the dominant mechanism for the maintenance of nuclear and cytoplasmic proteomes.
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Figure 1. Quantification of Nucleocytoplasmic Partitioning of the X. laevis Oocyte Proteome(A) Oocytes were dissected manually in three replicates, proteins digested, TMT-labeled and analyzed separately, with two different methods of accurate quantitative proteomics (MultiNotch MS3 and TMTC).(B) The relative nuclear concentration (RNC) was determined for 9,262 proteins. The replicates correlated with an R2 of at least 0.94.(C) RNC histogram of all quantified proteins.(D) Histogram of RNC values for proteins matched with the human MitoCarta database.(E) RNC histogram for proteins classified as nuclear within four commonly used subcellular localization databases are highly enriched for truly nuclear proteins (pink). However, the individual databases show only moderate agreement among themselves and with our data.See also Figures S1, S2, and S4.
Figure 2. Correlation of Molecular Weight and Nucleocytoplasmic Partitioning(A) Polypeptide molecular weight is not a strong determinant of nucleocytoplasmic distribution.(B) For estimation of native protein sizes, cell lysate was percolated through filters of 30 or 100 kDa molecular weight cutoff, respectively. The proteins’ relative passage was quantified with the MultiNotch MS3 approach.(C) Ratios of input and flow-through of the indicated filters were plotted and fitted with a spline. Color code and data point size indicate polypeptide molecular weight. Data point projection onto the spline yielded a “proxy for protein size,” ranging from 0 (small; bottom left) to 1 (large; top right).(D) This “proxy for protein size” and the experimentally determined native molecular weight for various vertebrate proteins correlate with an R2 of 0.95. This relationship allowed us to regress the native proteins size in a proteome-wide fashion.(E) Plot of native molecular weight versus polypeptide molecular weight indicates that many proteins behave significantly larger than their polypeptide molecular weight suggests. The few proteins for which we measured smaller native molecular weight than polypeptide molecular weight most likely represent measurement errors.(F) Histogram relating native molecular weight and RNC. Proteins smaller than ∼100 kDa are preferentially equipartitioned, whereas partitioned proteins are typically larger. However, a subset of natively large proteins is close to equipartitioned. Among them, we found the proteasome and APC/C.(G) Plot of estimated concentrations and RNCs for the subunits of the proteasome and the APC/C. Interestingly, the 19S and 11S α,β caps are slightly more nuclear than the core proteasome. In contrast, the 11S γ cap is exclusively nuclear.
Figure 3. Nucleocytoplasmic Protein Partitioning upon Inhibition of Exportin 1(A) Experimental setup to determine the change of RNCs upon inhibition of Exportin 1 with LMB.(B) RNCs determined for control oocytes and oocytes treated with LMB (24 hr) were plotted (experiment 1). The majority of proteins did not change its localization significantly (97%). Three proteins, which re-localized to the nucleus, are highlighted for illustration.(C) Scatter plot of RNC changes after 24 hr in LMB for (experiments 1 and 2). Under the assumption of noise being symmetric and LMB causing nuclear, but not cytoplasmic re-localization, we could estimate the FDR of LMB responders. With an FDR cutoff of ∼1% (dotted lines), we detected 226 confident LMB responders.(D) RNCs for all time points and replicates for the three highlighted proteins.(E) Most subunits of the APC/C responded to LMB, suggesting that at least some large complexes present in nucleus and cytoplasm (Figure 2F) are equipartitioned via active bidirectional transport. We did not see any evidence for Exportin 1-dependent nuclear transport of the proteasome.(F) Kinases are overrepresented among LMB responders (p = 0.002). The diagram shows these kinases.See also Figure S3.
Figure 4. The Maintenance of Nucleocytoplasmic Partitioning Is Dominated by Passive RetentionNuclear pores (depicted as holes in the nuclear envelope) permit the passage of small molecules but restrict that of larger ones. We observed that the vast majority of proteins smaller than ∼100 kDa (small green circles) have similar concentrations in nucleus and cytoplasm. Diffusion through nuclear pores allows these proteins to equilibrate between nucleus and cytoplasm. Nearly all partitioned proteins (red or blue) have a native molecular weight larger than ∼100 kDa, which prevents efficient diffusion through nuclear pores. Only very few natively small proteins are partitioned via continuous active transport. We also find a subset of natively large but equipartitioned proteins (large green circles). For some of these, we provide evidence that they are equilibrated by active bidirectional transport.
Update on activities at the Universal Protein Resource (UniProt) in 2013.