The Nuclear Proteome of a Vertebrate

Abstract

Summary 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 ~9000 proteins by mass spectrometry. Most proteins localize entirely to either nucleus or cytoplasm, 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, while 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 towards 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.

Landscape of nuclear transport receptor cargo specificity

“…On the other hand, GO terms enriched in the background proteome were mainly associated with functions typical of mitochondrial proteins (Fig 3B). We next compared our results with previous data of nucleocytoplasmic partitioning (Wühr et al , 2015), where each protein was assigned to the RNC (relative nuclear concentration) calculated for over 9,000 proteins of Xenopus laevis (Fig 3C). We found that the NIP shows a significant enrichment of proteins with high RNC values, indicative of asymmetric distribution with higher nuclear concentration.…”

“…Comparison of cargos known from literature for IPO5, KPNB1 (Kimura et al , 2017), and XPO1 (Thakar et al , 2013; Kırlı et al , 2015; Wühr et al , 2015). For XPO1, only cargos significant in at least two out of the three previous large‐scale studies were considered.…”

“…These include the interaction between XPO1 and snurportin‐1 (Paraskeva et al , 1999), the interaction between NXF1 and NXTs (Wiegand et al , 2002), and the reciprocal specificity between XPO2 and multiple of importin α (Fig 4G and Appendix Fig S2B). We also compared cargos identified in large‐scale studies for several importin βs (Fig 4H and Appendix Fig S2C; Kimura et al , 2017) and XPO1 (Fig 4I; Thakar et al , 2013; Kırlı et al , 2015; Wühr et al , 2015). The three large‐scale studies performed for XPO1 had limited overlap with each other in terms of identified cargos (maximum 29%).…”

“…Previous studies of XPO1 relied on a very potent inhibitor (leptomycin B; Thakar et al , 2013; Wühr et al , 2015), or the fact that export complexes are formed in the presences of RAN‐GTP for cargo identification (Kırlı et al , 2015). Cross‐validation of the cargos identified in our study against previous work on XPO1 showed statistically significant, but incomplete overlap (Fig 4I).…”

“…It remains understudied to which extent the functions of the different importins, exportins, and transportins are distinct or functionally redundant and to which extent the nuclear transport system contributes to protein localization. Experiments with the XPO1 inhibitor leptomycin B suggested that only a small fraction of the nuclear proteome is actively exported in Xenopus oocytes (Wühr et al , 2015), while aforementioned biochemical study has identified a rather large number of XPO1 cargos (Kırlı et al , 2015). Whether these results are generically applicable to somatic cells or specific to the very special cell properties of oocytes remains to be further explored.…”