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Maturation kinetics of a multiprotein complex revealed by metabolic labeling

ABSTRACT: The project aims at computing in vivo co-assembly kinetics from metabolomic-inspired approaches. In short, we performed reciprocal pull-down experiments from complex cellular components at different time points following pulsed-SILAC metabolic labeling. We applied the method to 320 pairs of yeast nucleoporin (NUP) proteins constituting the ~ 50 MDa Nuclear Pore Complex (NPC). Our analysis reveals a hierarchical principle of the NPC biogenesis where individual subcomplexes form on a minute time-scale, followed by their co-assembly from central to peripheral subunits in a ~ 1 hour long maturation process.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Ludovic Gillet

LAB HEAD: Evgeny Onischenko

PROVIDER: PXD018034 | Pride | 2020-10-16

REPOSITORIES: Pride

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			Action	DRS
	180912_LG_Evgeny81_1.raw	Raw
	180912_LG_Evgeny81_10b.raw	Raw
	180912_LG_Evgeny81_11.raw	Raw
	180912_LG_Evgeny81_12.raw	Raw
	180912_LG_Evgeny81_13.raw	Raw

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VAPB interacts with phosphorylated ELYS at decondensing chromosomes during mitosis (AP-MS Data)

Project description:ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate post-mitotic NPC assembly. Here we identified and characterized ELYS as a major binding partner of the membrane protein VAPB during mitosis. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which is shown to mediate interaction with the MSP (major sperm protein)-domain of VAPB. Phosphorylation-dependent binding of VAPB to ELYS is demonstrated by peptide binding assays and co-immunoprecipitation experiments. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB resulted in prolonged mitosis and slow progression from meta- to anaphase and also to chromosome segregation defects. Together, our results suggest a role of ELYS-VAPB-complexes in recruiting membrane fragments to chromatin.

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Effect of truncation mutants of the trypanosomatid nuclear lamina protein NUP-1 on the whole cell proteome in T. brucei

Project description:The nuclear lamina has multiple functions, including maintaining nuclear structural integrity and differential gene expression. Correct spatial and temporal lamina assembly is essential to meet these and other roles. Recently, it emerged that multiple lamina systems exist that are likely products of independent origins, while all these systems share remarkably analogous functions. Several lamina proteins are known in trypanosomes, two of which, NUP-1 and NUP-2, are essential, coiled-coil proteins with a molecular mass 450 and 250 kDa, respectively. Sequence analysis indicates distinct quaternary structures when compared to the ~60 kDa lamin proteins of multiple lineages, including metazoa. To uncover organisational principles of the trypanosome lamina we generated NUP-1 deletion mutants (N=N-terminal domain; C= C-terminal domain; NC: fusion of the N- and C-terminal domain with entire repeat region deletedd))designed to identify domains of NUP-1 responsible for oligomerisation. We find that both N- and C-termini act as interaction domains and disruption of these interactions impacts additional components of the lamina, the nuclear envelope and nucleoporin TbNup98. By contrast there is remarkably little impact on transcription, crucially including silencing of telomeric variant surface glycoprotein genes. These data indicate that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and suggest an architecture distinct to the lamin system is based on a ‘hub and spoke’ configuration.

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An Amphipathic Helix in Brl1 is Required for Nuclear Pore Complex Biogenesis in S. cerevisiae - KARMA analysis of Brl1 (DIA)

Project description:This project was aimed at analyzing the binding of Brl1 to the nuclear pore complex during the assembly process using the Kinetic Analysis of incorporation Rates in Macromolecular Assemblies (KARMA, Onischenko et al. 2020) method. In brief, we affinity purified Brl1 at several time points following SILAC metabolic labeling in growing S. cerevisiae cultures. Then, we analyzed the metabolic labeling for all co-purified nucleoporins and used these labeling kinetics to infer the timing of Brl1 binding during the native nuclear pore complex assembly.

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Y-complex nucleoporins independently contribute to nuclear pore assembly and gene regulation in neuronal progenitors

Project description:From their essential function in building up the nuclear pore complexes, nucleoporins have expanded roles beyond nuclear transport. Hence, their contribution to chromatin organization and gene expression has set them as critical players in development and pathologies. We previously reported that Nup133 and Seh1, two components of the Y-complex subunit of the nuclear pore scaffold, are dispensable in mouse embryonic stem cells but required for their survival during neuroectodermal differentiation. Here, a transcriptomic analysis revealed that Nup133 regulates a subset of genes at early stages of neuroectodermal differentiation, including Lhx1 and Nup210L, encoding a newly validated nucleoporin. These genes were also misregulated in Nup133∆Mid neuronal progenitors, in which NPC basket assembly is impaired, as previously observed in pluripotent cells. However, a four-fold reduction of Nup133, despite affecting basket assembly, is not sufficient to alter Nup210L and Lhx1 regulation. Finally, these two genes are misregulated in Seh1-deficient progenitors that only show a mild decrease in NPC density. Together these data reveal a shared function of Y-complex nucleoporins in gene regulation during neuroectodermal differentiation.

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Project description:This project was aimed at investigating nuclear pore complex biogenesis upon inducible degradation of Brl1 using the Kinetic Analysis of incorporation Rates in Macromolecular Assemblies (KARMA, Onischenko et al. 2020) method. In brief, the nuclear pore complex was purified through endogenously tagged Nup170 at several time points following auxin inducible degradation of Brl1. Simultaneously with the degradation of Brl1, newly synthesized proteins were metabolically labeled using a SILAC approach. The metabolic labeling of all nuclear pore complex constituents was assessed. Proteins that depend on Brl1 for their assembly show delayed metabolic labeling.

2022-08-17 | PXD032024 | Pride

Seh1 Interacts with Olig2 and Brd7 to Promote Oligodendrocyte Differentiation and Myelination (ATAC_Seq)

Project description:Nuclear pore complex components (Nups) are involved in neural development and alterations in Nup genes are linked to human neurological diseases. However, the physiological functions of specific Nups and the underlying mechanisms involved in these processes remain elusive. Here we show that tissue-specific depletion of nucleoporin Seh1 causes dramatic myelination defects in the Central Nervous System (CNS). Seh1-deficient Oligodendrocyte Progenitor Cells (OPCs) proliferate properly, but fail to differentiate into mature oligodendrocytes, which impairs myelin production and remyelination after demyelinating injury. Genome-wide analyses show that Seh1 regulates a core myelinogenic regulatory network and depletion of Seh1 alters open chromatin configurations at its target genes. Mechanistically, Seh1 regulates OPCs differentiation by assembling Olig2 and Brd7 into a transcription complex at nuclear pores. Together, our results reveal that Seh1 is required for oligodendrocyte differentiation and myelination by promoting assembly of an Olig2-dependent transcription complex and expose nucleoporins as key players in the CNS.

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Seh1 Interacts with Olig2 and Brd7 to Promote Oligodendrocyte Differentiation and Myelination (RNA-Seq)

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Probing the trypanosome nuclear pore by TurboID proximity labelling – Part2

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Nuclear pore complex evolution: A trypanosome Mlp analog functions in chromosomal segregation but lacks transcriptional barrier activity

Project description:The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organisation. In many eukaryotes the coiled coil Mlp/Tpr proteins of the NPC nuclear basket have specific roles in interactions with chromatin and defining specialised regions of active transcription, while Mlp2 associates with the mitotic spindle in a cell-cycle dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and low fidelity telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site, but apparently has minimal roles in the control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analog of Mlp/Tpr proteins, and together with the lamina analog NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina. Whole transcriptome comparison between parental and TbNup92? cells

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